PAN-CORONAVIRUS VACCINE COMPOSITIONS
20230226173 · 2023-07-20
Inventors
Cpc classification
A61K39/215
HUMAN NECESSITIES
C12N2770/20034
CHEMISTRY; METALLURGY
A61K2039/57
HUMAN NECESSITIES
A61K2039/545
HUMAN NECESSITIES
C07K14/165
CHEMISTRY; METALLURGY
International classification
Abstract
Pan-coronavirus recombinant vaccine compositions featuring whole proteins or sequences of proteins encompassing all mutations in variants of human and animal Coronaviruses (e.g., 36 mutations in spike protein) or a combination of mutated B cell epitopes, mutated combination of B cell epitopes, mutated CD4+ T cell epitopes, and mutated CD8+ T cell epitopes, at least one of which is derived from a non-spike protein. The mutated epitopes may comprise one or more mutations. The present invention also describes using several immuno-informatics and sequence alignment approaches to identify several human B cell, CD4+ and CD8+ T cell epitopes that are highly mutated. The vaccine compositions herein have the potential to provide long-lasting B and T cell immunity regardless of human and animal Coronaviruses mutations.
Claims
1.-514. (canceled)
515. A coronavirus recombinant vaccine composition, the composition comprising at least two of: a. one or more coronavirus B-cell target epitopes; b. one or more coronavirus CD4.sup.+ T cell target epitopes; c. one or more coronavirus CD8.sup.+ T cell target epitopes; wherein at least one epitope has a mutation as compared to its corresponding epitope in SARS-CoV-2 isolate Wuhan-Hu-1, wherein the epitopes are derived from a human coronavirus, an animal coronavirus, or a combination thereof; wherein at least one epitope is derived from a non-spike protein.
516. The composition of claim 515, wherein the human coronavirus is SARS-CoV-2 original strain or a SARS-CoV-2 variant and wherein the animal coronavirus is a bat coronavirus, a pangolin coronavirus, a civet cat coronavirus, a mink coronavirus, a camel coronavirus, or a coronavirus from another animal susceptible to coronavirus infection.
517. The composition of claim 515, wherein non-spike proteins are encoded by ORF1ab, ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF10, or Envelope protein, Membrane protein, Nucleocapsid protein.
518. The composition of claim 515, wherein one or more of the at least two target epitopes is in the form of a large sequence, wherein the large sequence is a whole protein expressed by SARS-CoV-2 or a SARS-CoV-2 variant, or derive from a partial protein sequence expressed by SARS-CoV-2 or a SARS-CoV-2 variant, or a combination thereof.
519. The composition of claim 518, wherein the large sequence is selected from a group consisting of SEQ ID NO: 143-151.
520. The composition of claim 515, wherein target epitopes are derived from a SARS-CoV-2 protein selected from a group consisting of: proteins encoded by ORF1ab, ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF10, or an Envelope protein, a Membrane protein, a Nucleocapsid protein, and a Spike protein.
521. The composition of claim 515, wherein the mutated epitopes are derived from one or more of: one or more SARS-CoV-2 human strains or variants in current circulation; one or more coronaviruses that have caused a previous human outbreak; one or more coronaviruses isolated from animals selected from a group consisting of bats, pangolins, civet cats, minks, camels, and other animals receptive to coronaviruses; or one or more coronaviruses that cause the common cold, wherein the one or more SARS-CoV-2 human strains or variants in current circulation are selected from: strain B.1.177; strain B.1.160, strain B.1.1.7; strain B.1.351; strain P.1; strain B.1.427/B.1.429; strain B.1.258; strain B.1.221; strain B.1.367; strain B.1.1.277; strain B.1.1.302; strain B.1.525; strain B.1.526, strain S:677H, and strain S:677P, and wherein the one or more coronaviruses that cause the common cold are selected from: 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, and HKU1 beta coronavirus.
522. The composition of claim 515, wherein the one or more coronavirus CD8+ T cell target epitopes are selected from: SEQ ID NO: 2-29, SEQ ID NO: 30-57, SEQ ID NO: 153, or a combination thereof, wherein the one or more coronavirus CD4+ T cell target epitopes are selected from: SEQ ID NO: 58-73, SEQ ID NO: 74-105, SEQ ID NO: 154, or a combination thereof, and wherein one or more coronavirus B-cell target epitopes are selected from: SEQ ID NO: 106-116, SEQ ID NO: 117-138, SEQ ID NO: 155, SEQ ID NO: 172-178, or a combination thereof.
523. The composition of claim 515, wherein the mutated epitope is in a spike (S) protein, wherein the mutation is one or a combination of A22V, S477N, H69-, V70-, Y144-, N501Y, A570D, P681H, D80A, D215G, L241-, L242-, A243-, K417N, E484K, N501Y, A701V, L18F, K417T, E484K, N501Y, H655Y, S13I, W152C, L452R, S439K, S98F, D80Y, A626S, V1122L, A67V, H69-, V70-, Y144-, E484K, Q677H, F888L, L5F, T95I, D253G, E484K, A701V, Q677H, or Q677P.
524. The composition of claim 515, wherein the mutated epitope is in a nucleocapsid (N) protein, wherein the mutation is one or a combination of A220V, M234I, A376T, R203K, G204R, T205I, P80R, R203K, G204R, P199L, S186Y, D377Y, S2-, D3Y, A12G, P199L, M234I, P67S, P199L, D377Y, P67S, or P199L.
525. The composition of claim 515, wherein the mutated epitope is in an Envelope (E) protein, wherein the mutation is P71L.
526. The composition of claim 515, wherein the mutated epitope is in a protein encoded by ORF3a, wherein the mutation is one or a combination of Q38R, G172R, V202L, or P42L.
527. The composition of claim 515, wherein the mutated epitope is in a protein encoded by ORF7a, wherein the mutation is R80I.
528. The composition of claim 515, wherein the mutated epitope is in a protein encoded by ORF8, wherein the mutation is Q27*, T11I, or a combination thereof.
529. The composition of claim 515, wherein the mutated epitope is in a protein encoded by ORF10, wherein the mutation is V30L.
530. The composition of claim 515, wherein the mutated epitope is in a protein encoded by ORF1b protein, wherein the mutation is one or a combination of A176S, V767L, K1141R, E1184D, D1183Y, P255T, Q1011H, N1653D, R2613C, N1653D, or R2613C.
531. The composition of claim 515, wherein the mutated epitope is in a protein encoded by ORF1a protein, wherein the mutation is one or a combination of S3675-, G3676-, F3677-, S3675-, G3676-, F3677-, S3675-, G3676-, F3677-, 14205V, I2501T, T945I, T1567I, Q3346K, V3475F, M3862I, S3675-, G3676-, F3677-, S3675-, G3676-, F3677-, T265I, L3352F, T265I, or L3352F.
532. The composition of claim 515 further comprising a T cell attracting chemokine, wherein the T cell attracting chemokine is CCL5, CXCL9, CXCL10, CXCL11, or a combination thereof.
533. The composition of claim 515 further comprising a composition that promotes T cell proliferation and T-cell memory, wherein the composition that promotes T cell proliferation and memory is IL-7, IL-2, or IL-15.
534. The composition of claim 515, wherein the composition comprises one of SEQ ID NO: 139-141.
Description
DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0128] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:
[0129]
[0130]
[0131]
[0132]
[0133]
[0134]
[0135]
[0136]
[0137]
[0138]
[0139]
[0140]
[0141]
[0142]
[0143]
[0144]
[0145]
[0146]
[0147]
[0148]
[0149]
[0150]
[0151]
[0152]
[0153]
[0154]
[0155]
[0156]
[0157]
[0158]
[0159]
[0160]
[0161]
[0162]
[0163]
[0164]
[0165]
[0166]
[0167]
[0168]
[0169]
[0170]
[0171]
[0172]
[0173]
[0174]
[0175]
[0176]
[0177]
[0178]
[0179]
[0180]
[0181]
[0182]
TERMS
[0183] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation. Stated another way, the term “comprising” means “including principally, but not necessary solely”. Furthermore, variation of the word “comprising”, such as “comprise” and “comprises”, have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not (“comprising”).
[0184] Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990: and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods In Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press. San Diego, Calif.). Culture of Animal Cells: A Manual of Basic Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ad. E. J. Murray, The Humana Press Inc., Clifton. N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.
[0185] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
[0186] As used herein, the terms “immunogenic protein, polypeptide, or peptide” or “antigen” refer to polypeptides or other molecules (or combinations of polypeptides and other molecules) that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. In embodiments, the protein fragment has substantially the same Immunological activity as the total protein. Thus, a protein fragment according to the disclosure can comprises or consists essentially of or consists of at least one epitope or antigenic determinant. An “immunogenic” protein or polypeptide, as used herein, may include the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. “Immunogenic fragment” refers to a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above.
[0187] Synthetic antigens are also included within the definition, for example, poly-epitopes, flanking epitopes, and other recombinant or synthetically derived antigens. Immunogenic fragments for purposes of the disclosure may feature at least about 1 amino acid, at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.
[0188] As used herein, the term “epitope” refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
[0189] As used herein, the term “immunological response” to a composition or vaccine refers to the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. The host may display either a therapeutic or protective immunological response so resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
[0190] As used herein, the term “variant” refers to a substantially similar sequence. For polynucleotides, a variant comprises a deletion and/or addition and/or change of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or an amino acid sequence, respectively. Variants of a particular polynucleotide of the disclosure (e.g., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. “Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they have the ability to elicit an immune response.
[0191] The HLA-DR/HLA-A*0201/hACE2 triple transgenic mouse model referred to herein is a novel susceptible animal model for pre-clinical testing of human COVID-19 vaccine candidates derived from crossing ACE2 transgenic mice with the unique HLA-DR/HLA-A*0201 double transgenic mice. ACE2 transgenic mice are a hACE2 transgenic mouse model expressing human ACE2 receptors in the lung, heart, kidney and intestine (Jackson Laboratory, Bar Harbor, Me.). The HLA-DR/HLA-A*0201 double transgenic mice are “humanized” HLA double transgenic mice expressing Human Leukocyte Antigen HLA-A*0201 class I and HLA DR*0101 class II in place of the corresponding mouse MHC molecules (which are knocked out). The HLA-A*0201 haplotype was chosen because it is highly represented (>50%) in the human population, regardless of race or ethnicity. The HLA-DR/HLA-A*0201/hACE2 triple transgenic mouse model is a “humanized” transgenic mouse model and has three advantages: (1) it is susceptible to human SARS-CoV2 infection; (2) it develops symptoms similar to those seen in COVID-19 in humans; and (3) it develops CD4.sup.+ T cells and CD8.sup.+ T cells response to human epitopes. The novel HLA-DR/HLA-A*0201/hACE2 triple transgenic mouse model of the present invention may be used in the pre-clinical testing of safety, immunogenicity and protective efficacy of the human multi-epitope COVID-19 vaccine candidates of the present invention.
[0192] As used herein, the terms “treat” or “treatment” or “treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow the development of the disease, such as slow down the development of a disorder, or reducing at least one adverse effect or symptom of a condition, disease or disorder, e.g., any disorder characterized by insufficient or undesired organ or tissue function. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is “effective” If the progression of a disease is reduced or halted. That is, “treatment” Includes not just the improvement of symptoms or decrease of markers of the disease, but also a cessation or slowing of progress or worsening of a symptom that would be expected in absence of treatment. Beneficial or desired clinical results Include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” also includes ameliorating a disease, lessening the severity of its complications, preventing it from manifesting, preventing it from recurring, merely preventing it from worsening, mitigating an inflammatory response included therein, or a therapeutic effort to affect any of the aforementioned, even if such therapeutic effort is ultimately unsuccessful.
[0193] As used herein, the term “carrier” or “pharmaceutically acceptable carrier” or “pharmaceutically acceptable vehicle” refers to any appropriate or useful carrier or vehicle for Introducing a composition to a subject. Pharmaceutically acceptable carriers or vehicles may be conventional but are not limited to conventional vehicles. For example, E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 15th Edition (1975) and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore Md. and Philadelphia, Pa., 21.sup.st Edition (2006) describe compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules. Carriers (e.g., pharmaceutical carriers, pharmaceutical vehicles, pharmaceutical compositions, pharmaceutical molecules, etc.) are materials generally known to deliver molecules, proteins, cells and/or drugs and/or other appropriate material into the body. In general, the nature of the carrier will depend on the nature of the composition being delivered as well as the particular mode of administration being employed. In addition to biologically-neutral carriers, pharmaceutical compositions administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like. Patents that describe pharmaceutical carriers include, but are not limited to: U.S. Pat. Nos. 6,667,371; 6,613,355; 6,596,296; 6,413,536; 5,968,543; 4,079,038; 4,093,709; 4,131,648; 4,138,344; 4,180,646; 4,304,767; 4,946,931, the disclosures of which are incorporated in their entirety by reference herein. The carrier may, for example, be solid, liquid (e.g., a solution), foam, a gel, the like, or a combination thereof. In some embodiments, the carrier comprises a biological matrix (e.g., biological fibers, etc.). In some embodiments, the carrier comprises a synthetic matrix (e.g., synthetic fibers, etc.). In certain embodiments, a portion of the carrier may comprise a biological matrix and a portion may comprise synthetic matrix.
[0194] As used herein “coronavirus” may refer to a group of related viruses such as but not limited to severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). All the coronaviruses cause respiratory tract infection that range from mild to lethal in mammals. Several non-limiting examples of Coronavirus strains are described herein. In some embodiments, the compositions may protect against any Sarbecoviruses including but not limited to SARS-CoV1 or SARS-CoV2.
[0195] As used herein, “severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)” is a betacoronavirus that causes Coronavirus Disease 19 (COVID-19).
[0196] A “subject” is an individual and includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird, a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included. A “patient” is a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects
[0197] The terms “administering”, and “administration” refer to methods of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, administering the compositions orally, parenterally (e.g., intravenously and subcutaneously), by intramuscular injection, by intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like.
[0198] A composition can also be administered by topical intranasal administration (intranasally) or administration by inhalant. As used herein, “topical intranasal administration” means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism (device) or droplet mechanism (device), or through aerosolization of the composition. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. As used herein, “an inhaler” can be a spraying device or a droplet device for delivering a composition comprising the vaccine composition, in a pharmaceutically acceptable carrier, to the nasal passages and the upper and/or lower respiratory tracts of a subject. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intratracheal intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular composition used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
[0199] A composition can also be administered by buccal delivery or by sublingual delivery. As used herein “buccal delivery” may refer to a method of administration in which the compound is delivered through the mucosal membranes lining the cheeks. In some embodiment, for a buccal delivery the vaccine composition is placed between the gum and the cheek of a patient. As used herein “sublingual delivery” may refer to a method of administration in which the compound is delivered through the mucosal membrane under the tongue. In some embodiments, for a sublingual delivery the vaccine composition is administered under the tongue of a patient.
[0200] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration Involves use of a slow release or sustained release system such that a constant dosage is maintained. See, for example, U.S. Pat. No. 3,610,795, which is Incorporated by reference herein.
DETAILED DESCRIPTION OF THE INVENTION
[0201] Before the present compounds, compositions, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or to specific compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.
Multi-Epitope Pan-Coronavirus Vaccines
[0202] The present invention features Coronavirus vaccine compositions, methods of use, and methods of producing said vaccines, methods of preventing coronavirus infections, etc. The present invention also provides methods of testing said vaccines, e.g., using particular animal models and clinical trials. The vaccine compositions herein can induce efficient and powerful protection against the coronavirus disease or infection, e.g., by inducing the production of antibodies (Abs), CD4.sup.+ T helper (Th1) cells, and CD.sup.+8 cytotoxic T-cells (CTL).
[0203] The vaccine compositions, e.g., the antigens, herein feature multiple epitopes, which helps provide multiple opportunities for the body to develop an immune response for preventing an Infection.
[0204] In certain embodiments, the epitopes comprise mutations from variant strains of human coronaviruses and/or animal coronaviruses (e.g., coronaviruses isolated from animals susceptible to coronavirus Infections). In other embodiments, the epitopes are highly mutated among human coronaviruses and/or animal coronaviruses (e.g., coronaviruses isolated from animals susceptible to coronavirus infections). The vaccines herein may be designed to be effective against past, current, and future coronavirus outbreaks.
[0205] The target epitopes may be derived from structural (e.g., spike glycoprotein, envelope protein, membrane protein, nucleoprotein) or non-structural proteins of the coronaviruses.
[0206] In some embodiments, the vaccine composition comprises one or more coronavirus B-cell target epitopes; one or more coronavirus CD4.sup.+ T cell target epitopes; and one or more coronavirus CD8.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus B-cell target epitopes and one or more coronavirus CD4.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus B-cell target epitopes and one or more coronavirus CD8.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus CD8.sup.+ target epitopes and one or more coronavirus CD4.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus CD8.sup.+ target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus CD4.sup.+ target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus B cell target epitopes.
[0207] In some embodiments, the vaccine composition comprises mutated target epitopes. In some embodiments, the vaccine composition comprises mutated target epitopes. In some embodiments, the vaccine composition comprises a combination of mutated and mutated target epitopes
[0208] As will be discussed herein, in certain embodiments, the vaccine composition comprises whole spike protein, one or more coronavirus CD4.sup.+ T cell target epitopes; and one or more coronavirus CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises at least a portion of the spike protein (e.g., wherein the portion comprises a trimerized SARS-CoV-2 receptor-binding domain (RBD)), one or more coronavirus CD4.sup.+ T cell target epitopes; and one or more coronavirus CD8.sup.+ T cell target epitopes.
[0209] In certain embodiments, the vaccine composition comprises one or more coronavirus B cell target epitopes, one or more coronavirus CD4 T cell target epitopes: and one or more coronavirus CD8.sup.+ T cell target epitopes. For example, in certain embodiments, the vaccine composition comprises 4 B cell target epitopes, 15 CD8.sup.+ T cell target epitopes, and 6 CD4.sup.+ T cell target epitopes. The present invention is not limited to said combination of epitopes.
[0210] In certain embodiments, the vaccine composition comprises 1-10 B cell target epitopes. In certain embodiments, the vaccine composition comprises 2-10 B cell target epitopes. In certain embodiments, the vaccine composition comprises 2-15 B cell target epitopes. In certain embodiments, the vaccine composition comprises 2-20 B cell target epitopes. In certain embodiments, the vaccine composition comprises 2-30 B cell target epitopes. In certain embodiments, the vaccine composition comprises 2-15 B cell target epitopes. In certain embodiments, the vaccine composition comprises 2-5 B cell target epitopes. In certain embodiments, the vaccine composition comprises 5-10 B cell target epitopes. In certain embodiments, the vaccine composition comprises 5-15 B cell target epitopes. In certain embodiments, the vaccine composition comprises 5-20 B cell target epitopes. In certain embodiments, the vaccine composition comprises 5-25 B cell target epitopes. In certain embodiments, the vaccine composition comprises 5-30 B cell target epitopes. In certain embodiments, the vaccine composition comprises 10-20 B cell target epitopes. In certain embodiments, the vaccine composition comprises 10-30 B cell target epitopes.
[0211] In certain embodiments, the vaccine composition comprises 1-10 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-10 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-15 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-20 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-30 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-15 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-5 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-10 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-15 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-20 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-25 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-30 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 10-20 CD8.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 10-30 CD8.sup.+ T cell target epitopes.
[0212] In certain embodiments, the vaccine composition comprises 1-10 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-10 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-15 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-20 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-30 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-15 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 2-5 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-10 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-15 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-20 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 5-25 CD4.sup.+ T cell target epitopes. In certain embodiments, the composition comprises 5-30 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 10-20 CD4.sup.+ T cell target epitopes. In certain embodiments, the vaccine composition comprises 10-30 CD4.sup.+ T cell target epitopes.
[0213] Table 1 below further describes various non-limiting combinations of numbers of CD4 T cell target epitopes, CD8.sup.+ T cell target epitopes, and B cell target epitopes. The present invention is not limited to the examples described herein. In some embodiments, the target epitopes may be mutated, mutated, or a combination thereof.
TABLE-US-00001 TABLE 1 # B Cell # CD8* T Cell # CD4* T Cell Example Epitopes Epitopes Epitopes 1 4 15 6 2 5 10 7 3 4 12 8 4 1 16 9 5 2 2 2 6 1 5 5 7 4 6 6 8 3 12 4 9 3 3 3 10 1 14 8 11 2 10 5 12 4 9 3 13 3 3 7 14 5 11 4 15 2 8 6 16 3 9 8 17 2 10 4 18 4 6 7 19 3 14 3 20 2 4 5
[0214] The epitopes may be each separated by a linker. In certain embodiments, the linker allows for an enzyme to cleave between the target epitopes. The present invention is not limited to particular linkers or particular lengths of linkers. As an example, in certain embodiments, one or more epitopes may be separated by a linker 2 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 3 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 4 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 5 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 6 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 7 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 8 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 9 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker 10 amino acids in length. In certain embodiments, one or more epitopes may be separated by a linker from 2 to 10 amino acids in length.
[0215] Linkers are well known to one of ordinary skill in the art. Non-limiting examples of linkers include AAY, KK, and GPGPG. For example, in certain embodiments, one or more CD8.sup.+ T cell epitopes are separated by AAY. In some embodiments, one or more CD4.sup.+ T cell epitopes are separated by GPGPG. In certain embodiments, one or more B cell epitopes are separated by KK. In certain embodiments, KK is a linker between a CD4.sup.+ T cell epitope and a B cell epitope. In certain embodiments, KK is a linker between a CD8.sup.+ T cell epitope and a B cell epitope. In certain embodiments, KK is a linker between a CD8.sup.+ T cell epitope and a CD4.sup.+ T cell epitope. In certain embodiments, AAY is a linker between a CD4 T cell epitope and a B cell epitope. In certain embodiments, AAY is a linker between a CD8.sup.+ T cell epitope and a B cell epitope. In certain embodiments, AAY is a linker between a CD8.sup.+ T cell epitope and a CD4.sup.+ T cell epitope. In certain embodiments, GPGPG is a linker between a CD4.sup.+ T cell epitope and a B cell epitope. In certain embodiments, GPGPG is a linker between a CD8.sup.+ T cell epitope and a B cell epitope. In certain embodiments, GPGPG is a linker between a CD8.sup.+ T cell epitope and a CD4 T cell epitope.
[0216] The target epitopes may be derived from structural proteins, non-structural proteins, or a combination thereof. For example, structural proteins may include spike proteins (S), envelope proteins (E), membrane proteins (M), or nucleoproteins (N).
[0217] In some embodiments, the target epitopes are derived from at least one SARS-CoV-2 protein. The SARS-CoV-2 proteins may include ORF1ab protein, Spike glycoprotein, ORF3a protein, Envelope protein, Membrane glycoprotein, ORF6 protein, ORF7a protein, ORF7b protein, ORF8 protein, Nucleocapsid protein, and ORF10 protein. The ORF1ab protein provides nonstructural proteins (Nsp) such as Nsp1, Nsp2, Nsp3 (Papain-like protease), Nsp4, Nsp5 (3C-like protease), Nsp6, Nsp7, Nsp8, Nsp9, Nsp10, Nsp11, Nsp12 (RNA polymerase), Nsp13 (5′ RNA triphosphatase enzyme), Nsp14 (guanosineN7-methyltransferase), Nsp15 (endoribonuclease), and Nsp16 (2′-O-ribose-methyltransferase).
[0218] The SARS-CoV-2 has a genome length of 29,903 base pairs (bps) ssRNA (SEQ ID NO: 1). Generally, the region between 266-21555 bps codes for ORF1ab polypeptide: the region between 21583-25384 bps codes for one of the structural proteins (spike protein or surface glycoprotein); the region between 25393-26220 bps codes for the ORF3a gene; the region between 26245-26472 bps codes for the envelope protein; the region between 26523-27191 codes for the membrane glycoprotein (or membrane protein); the region between 27202-27387 bps codes for the ORF6 gene; the region between 27394-27759 bps codes for the ORF7a gene; the region between 27894-28259 bps codes for the ORF8 gene; the region between 28274-29533 bps codes for the nucleocapsid phosphoprotein (or the nucleocapsid protein); and the region between 29558-29674 bps codes for the ORF10 gene.
[0219] The one or more CD8.sup.+ T cell target epitopes may be derived from a protein selected from: spike glycoprotein. Envelope protein, ORF1ab protein, ORF7a protein, ORF8a protein, ORF10 protein, or a combination thereof. The one or more CD4.sup.+ T cell target epitopes may be derived from a protein selected from: spike glycoprotein, Envelope protein, Membrane protein, Nucleocapsid protein. ORF1a protein, ORF1ab protein, ORF6 protein, ORF7a protein, ORF7b protein, ORF8 protein, or a combination thereof. The one or more B cell target epitopes may be derived from the spike protein.
Mutations
[0220] The present invention features a coronavirus vaccine composition. In some embodiments, the composition comprises at least two of: one or more coronavirus B cell target epitopes, one or more coronavirus CD4+ T cell target epitopes; or one or more coronavirus CD8+ T cell target epitopes. In some embodiments, the epitopes are derived from a human coronavirus, an animal coronavirus, or a combination thereof. In certain embodiments, at least one of the epitopes is derived from a non-spike protein. In certain embodiments the composition induced immunity only to the epitopes.
[0221] For example, the present invention features pan-coronavirus recombinant vaccine compositions featuring whole proteins or sequences of proteins encompassing all mutations in variants of human and animal Coronaviruses (e.g., 38 mutations in spike protein shown in
[0222] In some embodiments, the human coronavirus is the SARS-CoV-2 original strain. e.g., SARS-CoV-2 isolate Wuhan-Hu-1. In some embodiments, the human coronavirus is a SARS-CoV-2 variant, such as but not limited to a variant of SARS-CoV-2 isolate Wuhan-Hu-1.
[0223] As used herein, “variant” may refer to a strain having one or more nucleic acid or amino acid mutations as compared to the original strain (such as but not limited to SARS-CoV-2 isolate Wuhan-Hu-1). In some embodiments, the SARS-CoV-2 variant epitope is derived from one or more of: strain B.1.177; strain B.1.180, strain B.1.1.7; strain B.1.351; strain P.1; strain B.1.427/8.1.429; strain B.1.258; strain B.1.221; strain B.1.387; strain B.1.1.277; strain B.1.1.302; strain B.1.525; strain B.1.526, strain S:677H, or strain S:877P.
[0224] In some embodiments, the animal coronavirus is a coronaviruses Isolated from animals selected from a group consisting of bats, pangolins, civet cats, minks, camels, and other animal receptive to coronaviruses.
[0225] Additionally, other coronaviruses may be used for determining mutated epitopes (including human SARS-CoVs as well as animal CoVs (e.g., bats, pangolins, civet cats, minks, camels, etc.)) that meet the criteria to be classified as “variants of concern” or “variants of interest.” Coronavirus variants that appear to meet one or more of the undermentioned criteria may be labeled “variants of interest” or “variants under investigation” pending verification and validation of these properties. In some embodiments, the criteria may include increased transmissibility, increased morbidity, increased mortality, increased risk of “long COVID”, ability to evade detection by diagnostic tests, decreased susceptibility to antiviral drugs (if and when such drugs are available), decreased susceptibility to neutralizing antibodies, either therapeutic (e.g., convalescent plasma or monoclonal antibodies) or in laboratory experiments, ability to evade natural immunity (e.g., causing reinfections), ability to infect vaccinated individuals, increased risk of particular conditions such as multisystem inflammatory syndrome or long-haul COVID or increased affinity for particular demographic or clinical groups, such as children or immunocompromised individuals. Once validated, variants of interest are renamed “variant of concern” by monitoring organizations, such as the CDC.
[0226] The vaccine composition may comprise mutated epitopes or large sequences. As used herein, the term “mutated” or “mutation” may refer to a change in one or more nucleic acids (or amino acids) as compared to the original sequence. In some embodiments, a nucleic acid mutation may be synonymous or non-synonymous.
[0227] In some embodiments, the epitope may comprise a D614G mutation, a T445C mutation, a C6288T mutation, a C26801G mutation, a C4543T mutation, a G5629T mutation, a C11497T mutation, a T26878C mutation, a C241T mutation, a C913T mutation, a C3037T mutation, a C5986T mutation, a C14678T mutation, a C15279T mutation, a T16176C mutation, a G174T mutation, a C241T mutation, a C3037T mutation, a C28253T mutation, a C241T mutation, a T733C mutation, a C2749T mutation, a C3037T mutation, a A6319G mutation, a A6813G mutation, a C12778T mutation, a C13860T mutation, a A28877T mutation, a G28878C mutation, a C2395T mutation, a T2597C mutation, a T24349C mutation, a G27890T mutation, a A28272T mutation, a C8047T mutation, a C28651T mutation, a G4980T mutation, a C6070T mutation, a C7303T mutation, a C7564T mutation, a C10279T mutation, a C10525T mutation, a C10582T mutation, a C27804T mutation, a C241T mutation, a C1498T mutation, a A1807G mutation, a G2659A mutation, a C3037T mutation, a T8593C mutation, a C9593T mutation, a C18171T mutation, a A20724G mutation, a C24748T mutation, a A28899G mutation, a G29543T mutation, a C241T mutation, a C3037T mutation, a A20262G mutation, a A28271- mutation, a C241T mutation, a G1942T mutation, a C3037T mutation, a A9085G mutation, a C14805T mutation, a C241T mutation, a C3037T mutation, a C21811A mutation, a T29194C mutation, a T29377 mutation, or combination thereof.
[0228] In some embodiments, the mutation may be a point mutation. In other embodiments, the mutation may be a single point mutation (such as the above mentioned mutations). In other embodiments, a single point mutation may be substitutions, deletions, or inversions.
[0229] In some embodiments, the mutations may be in any of the SARS-CoV-2 proteins which may include ORF1ab protein, Spike glycoprotein, ORF3a protein, Envelope protein, Membrane glycoprotein, ORF6 protein, ORF7a protein, ORF7b protein, ORF8 protein, Nucleocapsid protein, or ORF10 protein.
[0230] In some embodiments, mutations in the spike (S) protein may include but are not limited to A22V, S477N, H69-, V70-, Y144-, N501Y, A570D, P681H, D80A, D215G, L241-, L242-, A243-, K417N, E484K, N501Y, A701V, L18F, K417T, E484K, N501Y, H855Y, S13I, W152C, L452R, S439K, S98F, D80Y, A626S, V1122L, A67V, H69-, V70-, Y144-, E484K, Q677H, F888L, L5F, T95I, D253G, E484K, A701V, Q677H, Q677P or a combination thereof (also see
[0231] As previously discussed, in some embodiments, the composition comprises spike protein or portion thereof. Non-limiting examples of spike proteins with and without mutations are listed in Table 2.
TABLE-US-00002 TABLE 2 SEQ ID Sequence: NO: SARS-CoV-like SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPF 143 Spike-S1-NTD FSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI 13 bp-304 bp FGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSW MESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNID GYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRS YLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCAL DPLSETKCTLK SARS-CoV-2 RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADY 144 Spike-S1-RBD SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP 319 bp-541 bp GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF CoV Spike S1- FNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVTRAGCLIGAEHVN 145 S2_S2 NSYECDIPIGAGICASYQTQTNRDPQTLEILDITPCSFGGVSVITPGT 543 bp-1,208 bp NTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQSPR RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSM TKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNT QEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKV TLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYT SALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQ KLIANQFNSAIGKIQDSLSSTASALGKLQDVNQNAQALNTLVKQLS SNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVV FLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQR NFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKY FKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQ spike glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS 146 with a mutation VLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF 682-RRAR-685 .fwdarw. ASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF 682-QQAQ-685 in LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG the S1-S2 NFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGI cleavage site NITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKY NENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVTRAGCLIGAEHVNNSYECDIPIGAGICASY QTQTNRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCT EVPVAIHADQLTPTWRVYSTGSNVFQSPQQAQSVASQSIIAYTMSL GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST ECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPI KDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCL GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR LDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFT TAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWL GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT spike glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS 147 with two proline VLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF substitutions ASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF (K986P, V987P) LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG NFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGI NITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKY NENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSN QVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSL GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST ECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPI KDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCL GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR LDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFT TAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWL GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT spike glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS 148 with four proline VLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF substitutions ASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF (F817P, A892P, LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG A899P, A942P) NFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGI NITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKY NENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVTRAGCLIGAEHVNNSYECDIPIGAGICASY QTQTNRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCT EVPVAIHADQLTPTWRVYSTGSNVFQSPRRARSVASQSIIAYTMSL GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST ECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPL KDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCL GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTPSALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR LDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVFLHVTYVPAQEKNFT TAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWL GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT spike glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS 149 with six proline VLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF substitutions ASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF (F817P, A892P, LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG A899P, A942P, NFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGI K986P, V987P) NITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKY NENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVTRAGCLIGAEHVNNSYECDIPIGAGICASY QTQTNRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCT EVPVAIHADQLTPTWRVYSTGSNVFQSPRRARSVASQSIIAYTMSL GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST ECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPI KDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCL GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTPSALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR LDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFT TAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWL GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT spike glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS 150 with six proline VLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF substitutions ASTEKSNIIRGWFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF (F817P, A892P, LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG A899P, A942P, NFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGI K986P, V987P) NITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKY and a 682-RRAR- NENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV 685 .fwdarw. 682-QQAQ- RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS 685 mutation FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVTRAGCLIGAEHVNNSYECDIPIGAGICASY QTQTNRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCT EVPVAIHADQLTPTWRVYSTGSNVFQSPQQAQSVASQSIIAYTMSL GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST ECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPI KDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCL GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTPSALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR LDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFT TAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWL GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT Spike Glycoprotein M F V F L V L L P L V S S Q C V N F T T R T Q L P P A Y T N S F T 151 sequence with 36 R G V Y Y P D K V F R S S V L H S T Q D L F L P F F S N V T W F mutations and 6 H A I - S G T N G T K R F D N P V L P F N D G V Y F A S T E K S deletions (-) N I I R G W I F G T T L D S K T Q S L L I V N N A T N V V I K V C E F Q F C N D P F L G V - Y H K N N K S W M E S E F R V Y S S A N N C T F E Y V S Q P F L M D L E G K Q G N F K N L R E F V F K N I D G Y F K I Y S K H T P I N L V R D L P Q G F S A L E P L V D L P I G I N I T R F Q T L - - - H R S Y L T P G D S S S G W T A G A A A Y Y V G Y L Q P R T F L L K Y N E N G T I T D A V D C A L D P L S E T K C T L K S F T V E K G I Y Q T S N F R V Q P T E S I V R F P N I T N L C P F S E I F N A T K F S S V Y A W D R R K I N N C V A D Y S F L Y N S A S F S T F K C Y G V S L N K L N D L C F T N V Y A D S F V I R G D Q V K Q I A P G Q T G N I A D Y N Y K L P D D F T G C V I A W N S K K L D S K V V G N H K Y R F R F - R K S N L K P F E R D I S T E I Y Q V G N K P C K G A K G L N C Y L P L K S Y G F Q P T Y G V G Y Q P H R V V V L S F E L L H A S A T V C G P K K S T N L V K N K C V N F N F N G L T G T G V L T E S N K K F L P F Q Q F G R D I A D T T D A V R D P Q T L E I L D I T P C S F G G V S V I T P G T N T S N Q V A V L Y Q D V N C T E V P V A I H A D Q L T P T W R V Y S T G S N V F Q T R A G C L I G A E H V N N S Y E C D I P I G A G I C A S Y Q T Q T N S P R R A R S V A S Q S I I A Y T M S L G A E N S V A Y S N N S I A I P T N F T I S V T T E I L P V S M T K T S V D C T M Y I C G D S T E C S N L L L Q Y G S F C T Q L N R A L T G I A V E Q D K N T Q E V F A Q V K Q I Y K T P P I K Y F G G F N F S Q I L P D P S K P S K R S F I E D L L F N K V T L A D A G F I K Q Y G D C L G D I A A R D L I C A Q K F N G L T V L P P L L T D E M I A Q Y T S A L L A G T I T S G W T F G A G A A L Q I P F A M Q M A Y R F N G I G V T Q N V L Y E N Q K L I A N Q F N S A I G K I Q D S L S S T A S A L G K L Q D V V N Q N A Q A L N T L V K Q L S S N F G A I S S V L N D I L S R L D K V E A E V Q I D R L I T G R L Q S L Q T Y V T Q Q L I R A A E I R A S A N L A A T K M S E C V L G Q S K R V D F C G K G Y H L M S F P Q S A P H G V V F L H V T Y V P A Q E K N F T T A P A I C H D G K A H F P R E G V F V S N G T H W F V T Q R N F Y E P Q I T T D N T F V S G N C D V V I G I V N N T V Y D P L Q P E L D S F K E E L D K Y F K N H T S P D V D L G D I S G I N A S V V N I Q K E I D R L N E V A K N L N E S L I D L Q E L G K Y E Q Y I K W P W Y I W L G F I A G L I A I V M V T I M L C C M T S C C S C L K G C C S C G S C C K F D E D D S E P V L K G V K L H Y T Wild type native MFVFLVLLPLVSS 63 leader sequence
[0232] In some embodiments, the mutations in the nucleocapsid (N) protein may include but are not limited to A220V, M234I, A376T, R203K, G204R, T205I, P80R, R203K, G204R, P199L, S186Y, D377Y, S2-, D3Y, A12G, P199L, M234I, P67S, P199L, D377Y, P67S, P199L or a combination thereof.
[0233] In some embodiments, the mutations in the Envelope (E) protein may include but are not limited to P71L. In some embodiments, the mutations in the ORF3a protein may Include but are not limited to Q38R, G172R, V202L, P42L or a combination thereof.
[0234] In some embodiments, the mutations in the ORF7a protein may include but are not limited to R80I. In some embodiments, the mutations in the ORF8 protein may Include but are not limited to Q27, T11I, or a combination thereof. In some embodiments, mutation in the ORF10 protein may Include but are not limited to V30L.
[0235] In some embodiments, the mutations in the ORF1b protein may include but are not limited to A176S, V767L, K1141R, E1184D, D1183Y, P255T, Q1011H, N1653D, R2613C, N1653D, or a combination thereof.
[0236] In some embodiments, the mutations in the ORF1a protein may Include but are not limited to S3675-, G3676-, F3677-, S3675-, G3676-, F3677-, S3675-, G3676-, F3677-, 14205V, I2501T, T945I, T15871, Q3346K, V3475F, M3862I, S3875-, G3678-, F3677-, S3675-, G3678-, F3677-, T2851, L3352F, T265I, L3352F or a combination thereof.
[0237] In some embodiments, the vaccine composition comprises one or more coronavirus B-cell target epitopes; one or more coronavirus CD4.sup.+ T cell target epitopes; and one or more coronavirus CD8.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus B-cell target epitopes and one or more coronavirus CD4.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus B-cell target epitopes and one or more coronavirus CD8.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus CD8.sup.+ target epitopes and one or more coronavirus CD4.sup.+ T cell target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus CD8.sup.+ target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus CD4.sup.+ target epitopes. In some embodiments, the vaccine composition comprises one or more coronavirus B cell target epitopes.
[0238] In some embodiments, the one or more of the at least two target epitopes may be in the form of a large sequence. In some embodiments, the large sequence is derived from one or more whole protein sequences expressed by SARS-CoV-2 or a SARS-CoV-2 variant. In other embodiments, the large sequence is derived from one or more partial protein sequences expressed by SARS-CoV-2 or a SARS-CoV-2 variant.
[0239] The target epitopes may be derived from structural proteins, non-structural proteins, or a combination thereof. For example, structural proteins may include spike proteins (S), envelope proteins (E), membrane proteins (M), or nucleoproteins (N).
[0240] In some embodiments, the target epitopes are derived from at least one SARS-CoV-2 protein. The SARS-CoV-2 proteins may include ORF1ab protein. Spike glycoprotein, ORF3a protein, Envelope protein, Membrane glycoprotein, ORF6 protein, ORF7a protein, ORF7b protein, ORF8 protein, Nucleocapsid protein, and ORF10 protein. The ORF1ab protein provides nonstructural proteins (Nsp) such as Nsp1, Nsp2, Nsp3 (Papain-like protease), Nsp4, Nsp5 (3C-like protease), Nsp6, Nsp7, Nsp8, Nsp9, Nsp10, Nsp11, Nsp12 (RNA polymerase), Nsp13 (5′ RNA triphosphatase enzyme), Nsp14 (guanosineN7-methyltransferase), Nsp15 (endoribonuclease), and Nsp16 (2′-O-ribose-methyltransferase).
[0241] The target epitopes may be restricted to human HLA class 1 and 2 haplotypes. In some embodiments, the target epitopes are restricted to cat and dog MHC class 1 and 2 haplotypes.
Conserved Epitopes
[0242] In certain embodiments, the vaccine composition comprises one or more mutated epitopes in combination with one or more mutated epitopes.
[0243] The present invention describes the identification of mutated B cell, CD4.sup.+ T cell, and CD8.sup.+ T cell epitopes. For example,
[0244] Coronaviruses used for determining mutated epitopes may include human SARS-CoVs as well as animal CoVs (e.g., bats, pangolins, civet cats, minks, camels, etc.) as described herein. As an example,
[0245] Additionally, other coronaviruses may be used for determining mutated epitopes (including human SARS-CoVs as well as animal CoVs (e.g., bats, pangolins, civet cats, minks, camels, etc.)) that meet the criteria to be classified as “variants of concern” or “variants of interest.” Coronavirus variants that appear to meet one or more of the undermentioned criteria may be labeled “variants of interest” or “variants under investigation” pending verification and validation of these properties. In some embodiments, the criteria may include increased transmissibility, increased morbidity, increased mortality, increased risk of “long COVID”, ability to evade detection by diagnostic tests, decreased susceptibility to antiviral drugs (if and when such drugs are available), decreased susceptibility to neutralizing antibodies, either therapeutic (e.g., convalescent plasma or monoclonal antibodies) or in laboratory experiments, ability to evade natural immunity (e.g., causing reinfections), ability to infect vaccinated individuals, increased risk of particular conditions such as multisystem inflammatory syndrome or long-haul COVID or Increased affinity for particular demographic or clinical groups, such as children or immunocompromised individuals. Once validated variants of interest are renamed “variant of concern” by monitoring organizations, such as the CDC.
[0246] The mutated epitopes may be derived from structural (e.g., spike glycoprotein, envelope protein, membrane protein, nucleoprotein) or non-structural proteins of the coronaviruses (e.g., any of the 16 NSPs encoded by ORF1a/b).
[0247] In some embodiments, one or more epitopes are highly mutated among one or a combination of: SARS-CoV-2 human strains, SL-CoVs isolated from bats, SL-CoVs isolated from pangolin, SL-CoVs isolated from civet cats, and MERS strains Isolated from camels. For example, in certain embodiments, an epitopes is highly mutated among one or a combination of: at least 50,000 SARS-CoV-2 human strains, five SL-CoVs isolated from bats, five SL-CoVs isolated from pangolin, three SL-CoVs isolated from civet table cats, and four MERS strains isolated from camels. In certain embodiments, one or more epitopes are highly mutated among one or a combination of: at least 80,000 SARS-CoV-2 human strains, five SL-CoVs isolated from bats, five SL-CoVs isolated from pangolin, three SL-CoVs isolated from civet cats, and four MERS strains isolated from camels. In certain embodiments, one or more epitopes are highly mutated among one or a combination of: at least 50,000 SARS-CoV-2 human strains in circulation during the COVI-19 pandemic, at least one CoV that caused a previous human outbreak, five SL-CoVs Isolated from bats, five SL-CoVs isolated from pangolin, three SL-CoVs Isolated from civet cats, and four MERS strains isolated from camels. In certain embodiments, one or more epitopes are highly mutated among at least 1 SARS-CoV-2 human strain in current circulation, at least one CoV that has caused a previous human outbreak, at least one SL-CoV isolated from bats, at least one SL-CoV isolated from pangolin, at least one SL-CoV isolated from civet cats, and at least one MERS strain isolated from camels. In certain embodiments, one or more epitopes are highly mutated among at least 1,000 SARS-CoV-2 human strains in current circulation, at least two CoVs that has caused a previous human outbreak, at least two SL-CoVs isolated from bats, at least two SL-CoVs isolated from pangolin, at least two SL-CoVs isolated from civet cats, and at least two MERS strains isolated from camels. In certain embodiments, one or more epitopes are highly mutated among one or a combination of: at least one SARS-CoV-2 human strain in current circulation, at least one CoV that has caused a previous human outbreak, at least one SL-CoV isolated from bats, at least one SL-CoV isolated from pangolin, at least one SL-CoV isolated from civet cats, and at least one MERS strain isolated from camels. The present invention is not limited to the aforementioned coronavirus strains that may be used to identify mutated epitopes.
[0248] In certain embodiments, one or more of the mutated epitopes are derived from one or more SARS-CoV-2 human strains or variants in current circulation; one or more coronaviruses that has caused a previous human outbreak; one or more coronaviruses isolated from animals selected from a group consisting of bats, pangolins, civet cats, minks, camels, and other animal receptive to coronaviruses; and/or one or more coronaviruses that cause the common cold. SARS-CoV-2 human strains and variants in current circulation may include the original SARS-CoV-2 strain (SARS-CoV-2 isolate Wuhan-Hu-1), and several variants of SARS-CoV-2 including but not limited to Spain strain B.1.177; Australia strain B.1.160, England strain B.1.1.7; South Africa strain B.1.351; Brazil strain P.1; California strain B.1.427/B.1.429; Scotland strain B.1.258; Belgium/Netherlands strain B.1.221; Norway/France strain B.1.367; Norway/Denmark.UK strain B.1.1.277; Sweden strain B.1.1.302; North America, Europe, Asia, Africa, and Australia strain B.1.525; and New York strain B.1.526. The present invention is not limited to the aforementioned variants of SARS-CoV-2 and encompasses variants identified in the future. The one or more coronaviruses that cause the common cold may include but are not limited to strains 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus).
[0249] As used herein, the term “mutated” refers to an epitope that is among the most highly mutated epitopes identified in a sequence alignment and analysis for its particular epitopes type (e.g., B cell, CD4 T cell, CD8 T cell). For example, the mutated epitopes may be the 5 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 10 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 15 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 20 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 25 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 30 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 40 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 50 most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 50% most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 60% most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 70% most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 80% most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 90% most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 95% most highly mutated epitopes identified (for the particular type of epitope). In some embodiments, the mutated epitopes may be the 99% most highly mutated epitopes identified (for the particular type of epitope). The present invention is not limited to the aforementioned thresholds.
[0250]
[0251] For certain embodiments herein, the epitopes that are selected may be those that achieve a particular score in a binding assay (for binding to an HLA molecule, for example.) For example, in some embodiments, the epitopes selected have an IC.sub.50 score of 250 or less in an ELISA binding assay (e.g., an ELISA binding assay specific for HLA-DR/peptide combination, HLA-A*0201/peptide combination, etc.), or the equivalent of the IC.sub.50 score of 250 or less in a different binding assay. Binding assays are well known to one of ordinary skill in the art.
[0252] The mutated epitopes may be restricted to human HLA class 1 and 2 haplotypes. In some embodiments, the mutated epitopes are restricted to cat and dog MHC class 1 and 2 haplotypes.
[0253] For any of the embodiments herein, the epitopes that are selected may be those that achieve a particular score in a binding assay (for binding to an HLA molecule, for example.) For example, in some embodiments, the epitopes selected have an IC.sub.50 score of 250 or less in an ELISA binding assay (e.g., an ELISA binding assay specific for HLA-DR/peptide combination, HLA-A*0201/peptide combination, etc.), or the equivalent of the IC.sub.50 score of 250 or less in a different binding assay. Binding assays are well known to one of ordinary skill in the art.
[0254]
[0255]
CD8+ Epitopes
[0256] The present invention features a plurality of CD8+ T cell epitopes which may comprise one or more mutations. In some embodiments, a mutation may be synonymous or non-synonymous. In some embodiments, the mutation may be a point mutation. In other embodiments, the mutation may be a single point mutation (such as the above mentioned mutations). In other embodiments, a single point mutation may be substitutions, deletions, or inversions
[0257] Table 3: below describes the sequences for the mutated epitope regions. Bolded amino acids Indicate amino acids that have been mutated when compared to the SARS-CoV-2-Wuhan (MN908947.3) strain.
TABLE-US-00003 SEQ ID CD8.sup.+ Epitope Sequence NO: S.sub.976-894 VLNDILARL 153
[0258] Examples of methods for identifying potential CD8+ T cell epitopes and screening conservancy of potential CD8+ T cell epitopes are described herein. The present invention is not limited to the particular software systems disclosed, and other software systems are accessible to one of ordinary skill in the art for such methods. The present invention is not limited to the specific haplotypes used herein. For example, one of ordinary skill in the art may select alternative molecules (e.g., HLA molecules) for molecular docking studies.
[0259]
[0260] From the analysis, 27 CD8+ T cell epitopes were selected as being highly mutated.
[0261]
[0262] The CD8.sup.+ T cell target epitopes discussed above include S.sub.2-10, S.sub.1220-1228, S.sub.1000-1008, S.sub.958-866, E.sub.20-28, ORF1ab.sub.1675-1683, ORF1ab.sub.2363-2371, ORF1ab.sub.3013-3021, ORF1ab.sub.3183-3191, ORF1ab.sub.5470-5478, ORF1ab.sub.6749-6757, ORF7b.sub.26-34, ORF8a.sub.73-81, ORF10.sub.3-11, and ORF10.sub.5-13.
TABLE-US-00004 TABLE 4 CD8.sup.+ T Cell Epitope SEQ ID CD8.sup.+ T Cell Epitope SEQ ID Epitope Sequence NO: Epitope Sequence NO: ORF1ab.sub.84-92 VMVELVAEL 2 S.sub.976-984 VLNDILSRL 16 ORF1ab.sub.1675-1683 YLATALLTL 3 S.sub.1000-1008 RLQSLQTYV 17 ORF1ab.sub.2210-2218 CLEASFNYL 4 S.sub.1220-1228 FIAGLIAIV 18 ORF1ab.sub.2363-2371 WLMWLIINL 5 E.sub.20-28 FLAFWVFLL 19 ORF1ab.sub.3013-3021 SLPGVFCGV 6 E.sub.26-34 FLLVTLAIL 20 ORF1ab.sub.3183-3191 FLLNKEMYL 7 E.sub.26-34 FLLNKEMYL 21 ORF1ab.sub.3732-3740 SMWALIISV 8 M.sub.52-60 IFLWLLWPV 22 ORF1ab.sub.4283-4291 YLASGGQPI 9 M.sub.89-97 GLMWLSYFI 23 ORF1ab.sub.5470-5478 KLSYGIATV 10 ORF6.sub.3-11 HLVDFQVTi 24 ORF1ab.sub.6419-6427 YLDAYNMMI 11 ORF7b.sub.26-34 IIFWFSLEL 25 ORF1ab.sub.6749-6757 LLLDDFVEI 12 ORF8a.sub.31-39 YVVDDPCPI 26 S.sub.2-10 FVFLVLLPL 13 ORF8a.sub.73-81 YIDIGNYTV 27 S.sub.691-699 SIIAYTMSL 14 ORF10.sub.3-11 YINVFAFPF 28 S.sub.958-986 ALNTLVKQL 15 ORF10.sub.5-13 NVFAFPFTL 29
[0263] The present invention is not limited to the aforementioned CD8.sup.+ T cell epitopes. For example, the present invention also Includes variants of the aforementioned CD8.sup.+ T cell epitopes, for example sequences wherein the aforementioned CD8.sup.+ T cell epitopes are truncated by one amino acid (examples shown below in Table 5).
TABLE-US-00005 TABLE 5 CD8.sup.+ T Cell Sequence with CD8.sup.+ T Cell Sequence with Epitope Single AA SEQ ID Epitope Single AA SEQ ID Origin: Truncation NO: Origin: Truncation NO: ORF1ab.sub.84-92 VMVELVAE 30 S.sub.976-984 VLNDILSR 44 ORF1ab.sub.1675-1683 LATALLTL 31 S.sub.1000-1008 LQSLQTYV 45 ORF1ab.sub.2210-2218 CLEASFNY 32 S.sub.1220-1228 FIAGLIAI 46 ORF1ab.sub.2363-2371 LMWLIINL 33 E.sub.20-28 LAFVVFLL 47 ORF1ab.sub.3013-2021 SLPGVFCG 34 E.sub.26-34 FLLVTLAL 48 ORF1ab.sub.3183-3191 LLNKEMYL 35 E.sub.26-34 LLNKEMYL 49 ORF1ab.sub.3732-3740 SMWALIIS 36 M.sub.52-60 IFLWLLWP 50 ORF1ab.sub.4283-4291 LASGGQPI 37 M.sub.89-97 LMWLSYFI 51 ORF1ab.sub.5470-5478 KLSYGIAT 38 ORF6.sub.3-11 HLVDFQVT 52 ORF1ab.sub.6419-6427 LDAYNMMI 39 ORF7b.sub.26-34 IFWFSLEL 53 ORF1ab.sub.6749-6757 LLLDDFVE 40 ORF8a.sub.31-39 YWVDOPCP 54 S.sub.2-10 VFLVLLPL 41 ORF8a.sub.73-81 IDIGNYTV 55 S.sub.691-699 SIIAYTMS 42 ORF10.sub.3-11 YINVFAFP 56 S.sub.958-966 LNTLVKQL 43 ORF10.sub.5-13 VFAFPFTI 57
[0264] The present invention is not limited to the aforementioned CD8.sup.+ T cell epitopes.
CD4+ Epitopes
[0265] The present invention features a plurality of CD4+ T cell epitopes which may comprise one or more mutations. In some embodiments, a mutation may be synonymous or non-synonymous. In some embodiments, the mutation may be a point mutation. In other embodiments, the mutation may be a single point mutation (such as the above-mentioned mutations). In other embodiments, a single point mutation may be substitutions, deletions, or inversions
[0266] Table 6: below describes the sequences for the mutated epitope regions. Bolded amino acids indicate amino acids that have been mutated when compared to the SARS-CoV-2-Wuhan (MN908947.3) strain.
TABLE-US-00006 SEQ ID CD4.sup.+ Epitope Sequence NO: S.sub.1-13 MFVFLVLLPLVSI 154
[0267] Examples of methods for identifying potential CD4+ T cell epitopes and screening conservancy of potential CD4+ T cell epitopes are described herein. The present invention is not limited to the particular software systems disclosed, and other software systems are accessible to one of ordinary skill in the art for such methods. The present invention is not limited to the specific haplotypes used herein. For example, one of ordinary skill in the art may select alternative molecules (e.g., HLA molecules) for molecular docking studies.
[0268]
[0269] From the analysis, 16 CD4+ T cell epitopes were selected as being highly mutated.
[0270]
[0271] The CD4.sup.+ T cell target epitopes discussed above include ORF1a.sub.1350-1365, ORF1ab.sub.5019-5033, ORF6.sub.12-26, ORF1ab.sub.6088-6102, ORF1ab.sub.6420-6434, ORF1a.sub.1801-1815, S.sub.1-13, E.sub.26-40, E.sub.20-34, M.sub.176-190, N.sub.368-403, ORF7a.sub.3-17, ORF7a.sub.1-15, ORF7b.sub.8-22, ORF7a.sub.98-112, and ORF8.sub.1-15.
TABLE-US-00007 TABLE 7 SEQ SEQ CD4.sup.+ T Cell ID CD4.sup.+ T Cell ID Epitope Epitope Sequence NO: Epitope Epitope Sequence NO: ORF1a.sub.1350-1365 KSAFYILPSIISNEK 58 M.sub.176-190 LSYYKLGASQRVAGD 66 ORF1a.sub.1801-1815 ESPFVMMSAPPAQYE 59 ORF6.sub.12-26 AEILLIIMRTFKVSI 67 ORF1ab.sub.5019-5033 PNMLRIMASLVLARK 60 ORF7a.sub.1-5 MKIILFLALITLATC 68 ORF1ab.sub.6088-6102 RIKVQMLSDTLKNL 61 ORF7a.sub.3-17 IIFLALITLATCEL 69 ORF1ab.sub.6420-6434 LDAYNMMISAGFSLW 62 ORF7a.sub.98-112 SPIFLIVAAIVFITL 70 S.sub.1-13 MFVFLVLLPLVSS 63 ORF7b.sub.8-22 DFYLCFLAFLLFLVL 71 E.sub.20-34 FLAFVVFLLVTLAIL 64 ORF8b.sub.1-15 MKFLVFLGIITTVAA 72 E.sub.26-40 FLLVTLAILTALRLC 65 N.sub.388-4031 KQQTVTLLPAADLDDF 73
[0272] The present invention is not limited to the aforementioned CD4.sup.+ T cell epitopes. For example, the present invention also includes variants of the aforementioned CD4.sup.+ T cell epitopes, for example sequences wherein the aforementioned CD4.sup.+ T cell epitopes are truncated by one or more amino acids or extended by one or more amino acids (examples shown below in Table 8).
TABLE-US-00008 TABLE 8 Sequence with SEQ CD4.sup.+ T Cell Sequence with SEQ CD4.sup.+ T Cell Single ID Epitope Single ID Epitope Origin AA Truncation NO: Origin AA Truncation NO: ORF1a.sub.1350-1365 KSAFYILPSIISNE 74 ORF1a.sub.1350-1365 SAFYILPSIISNEK 90 ORF1a.sub.1801-1815 ESPFVMMSAPPAQY 75 ORF1a.sub.1801-1815 SPFVMMSAPPAQYE 91 ORF1ab.sub.5019-5033 PNMLRIMASLVLAR 76 ORF1ab.sub.5019-5033 NMLRIMASLVLARK 92 ORF1ab.sub.6088-6102 RIKVQMLSDTLKN 77 QRF1ab.sub.6086-6102 IKVOMLSDTLKNL 93 ORF1ab.sub.6420-6434 LDAYNMMISAGFSL 78 ORF1ab.sub.6420-6434 DAYNMMISAGFSLW 94 S.sub.1-13 MFVFLVLLPLVS 79 S.sub.1-13 FVFLVLLPLVSS 95 E.sub.20-34 FLAFVVFLLVTLAL 86 E.sub.20-34 LAFVVFLLVTLAIL 96 E.sub.26-40 FLLVTLAILTALRL 81 E.sub.26-40 LLVTLAILTALRLC 97 M.sub.176-190 LSYYKLGASQRVAG 82 M.sub.176-190 SYYKLGASQRVAGD 98 ORF6.sub.12-26 AEILLIIMRTFKVS 83 ORF6.sub.12-26 EILLIIMRTFKVS 99 ORF7a.sub.1-15 MKIILFLALITLAT 84 ORF7a.sub.1-15 KIILFLALITLATC 160 ORF7a.sub.3-17 IIFLALITLATCE 85 ORF7a.sub.3-17 IFLALITLATCEL 101 ORF7a.sub.96-112 SPIFLIVAAIVFIT 86 ORF7a.sub.96-112 PIFLIVAAIVFITL 102 ORF7b.sub.8-22 DFYLCFLAFLLFLV 87 ORF7b.sub.8-22 FYLCFLAFLLFLVL 103 ORF8b.sub.1-15 MIKFLVFLGIITTVA 88 ORF8b.sub.1-15 KFLVFLGIITTVAA 104 N.sub.388-4031 KQQTVTLLPAADLDD 89 N.sub.388-4031 QQTVTLLPAADLDDF 105
[0273] The present invention is not limited to the aforementioned CD4.sup.+ T cell epitopes.
B Cell Epitopes
[0274] The present invention features a plurality of B cell epitopes which may comprise one or more mutations. In some embodiments, a mutation may be synonymous or non-synonymous. In some embodiments, the mutation may be a point mutation. In other embodiments, the mutation may be a single point mutation (such as the above mentioned mutations). In other embodiments, a single point mutation may be substitutions, deletions, or Inversions.
[0275] Table 9: below describes the sequences for the mutated epitope regions. Bolded amino acids indicate amino acids that have been mutated when compared to the SARS-CoV-2-Wuhan (MN908947.3) strain.
TABLE-US-00009 B Cell SEQ ID Epitope Sequence NO: S.sub.13-37 IQCVNLTTRTQLPPAYTNSFTRGVY 155 S.sub.59-81 FSNVTWFHAIHVSGTNGTKRFAN 172 S.sub.287-317 DAVDCALDPLSETKCTLKSFTVEKGIYQTSN 173 S.sub.440-501 NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNC 174 YFPLQSYGFQPTY S.sub.440-501 NLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNC 175 YFPLQSYGFQPTE S.sub.524-598 VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIDDT 176 TDAVRDPQTLEILDITPCSFGGVSVI S.sub.601-640 GTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGS 177 S.sub.1133-1172 VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI 178
[0276] The present invention is not limited to the aforementioned B cell epitopes. For example, the present invention may also include other variants of the aforementioned B cell epitopes.
[0277] Examples of methods for identifying potential B cell epitopes and screening conservancy of potential B cell epitopes are described herein. The present invention is not limited to the particular software systems disclosed, and other software systems are accessible to one of ordinary skill in the art for such methods.
[0278]
[0279] From the analysis, 22 B cell epitopes were selected as being highly mutated.
[0280] The B cell target epitopes discussed above include S.sub.287-317, S.sub.524-598, S.sub.801-640, S.sub.802-819, S.sub.888-909, S.sub.369-393, S.sub.440-501, S.sub.1133-1172, S.sub.329-363, S.sub.59-81, and S.sub.13-37.
TABLE-US-00010 TABLE 10 B Cell SEQ ID Epitope Epitope Sequence NO: S.sub.13-37 SQCVNLTTRTQLPPAYTNSFTRGVY 106 S.sub.59-81 FSNVTWFHAIHVSGTNGTKRFDN 107 S.sub.287-317 DAVDCALDPLSETKCTLKSFTVEKGIYQTSN 108 S.sub.601-640 GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS 109 S.sub.524-598 VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDI 110 ADTTDAVRDPQTLEILDITPCSFGGVSVI S.sub.440-501 NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG 111 FNCYFPLQSYGFQPTE S.sub.369-393 YNSASFSTFKCYGVSPTKLNDLCFT 112 S.sub.329-363 FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA 113 S.sub.1133-1172 VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI 114 S.sub.802-819 FSQILPDPSKPSKRSFIE 115 S.sub.888-909 FGAGAALQIPFAMQMAYRFNGI 116
[0281] The present invention is not limited to the aforementioned B cell epitopes. For example, the present invention also includes variants of the aforementioned B cell epitopes, for example sequences wherein the aforementioned B cell epitopes are truncated by one or more amino acids or extended by one or more amino acids (examples shown below in Table 11).
TABLE-US-00011 TABLE 11 Origin of SEQ ID Epitope Sequence with AA Truncation NO: S.sub.13-35 SQCVNLTTRTQLPPAYTNSFTRG 117 S.sub.59-79 FSNVTWFHAIHVSGTNGTKRF 118 S.sub.287-315 DAVDCALDPLSETKCTLKSFTVEKGIYQT 119 S.sub.601-638 GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYST 120 S.sub.524-596 VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDI 121 ADTTDAVRDPQTLEILDITPCSFGGVS S.sub.440-499 NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG 122 FNCYFPLQSYGFQP S.sub.369-391 YNSASFSTFKCYGVSPTKLNDLC 123 S.sub.329-361 FPNITNLCPFGEVFNATRFASVYAWNRKRISNC 124 S.sub.1133-1170 VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS 12S S.sub.802-817 FSQILPDPSKPSKRSF 126 S.sub.888-907 FGAGAALQIPFAMQMAYRFN 127 S.sub.15-37 CVNLTTRTQLPPAYTNSFTRGVY 128 S.sub.61-81 NVTWFHAIHVSGTNGTKRFDN 129 S.sub.289-317 VDCALDPLSETKCTLKSFTVEKGIYQTSN 130 S.sub.603-640 NTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS 131 S.sub.526-598 GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIAD 132 TTDAVRDPQTLEILDITPCSFGGVSVI S.sub.442-501 DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN 133 CYFPLQSYGFQPTE S.sub.371-393 SASFSTFKCYGVSPTKLNDLCFT 134 S.sub.331-363 NITNLCPFGEVFNATRFASVYAWNRKRISNCVA 135 S.sub.1135-1172 NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI 136 S.sub.804-819 QILPDPSKPSKRSFIE 137 S.sub.890-909 AGAALQIPFAMQMAYRFNGI 138
[0282] As previously discussed, in some embodiments, the B cell epitope is in the form of whole spike protein. In some embodiments, the B cell epitope is in the form of a portion of spike protein. In some embodiments, the transmembrane anchor of the spike protein has an intact S1-S2 cleavage site. In some embodiments, the spike protein is in its stabilized conformation. In some embodiments, the spike protein is stabilized with proline substitutions at amino acid positions 988 and 987 at the top of the central helix in the S2 subunit. In some embodiments, the composition comprises a trimerized SARS-CoV-2 receptor-binding domain (RBD). In some embodiments, the trimerized SARS-CoV-2 receptor-binding domain (RBD) sequence is modified by the addition of a T4 fibritin-derived foldon trimerization domain. In some embodiments, the addition of a T4 fibritin-derived foldon trimerization domain Increases immunogenicity by multivalent display.
[0283] In some embodiments, the spike protein comprises Tyr-489 and Asn-487 (e.g., Tyr-489 and Asn-487 help with interaction with Tyr 83 and Gln-24 on ACE-2). In some embodiments, the spike protein comprises Gln-493 (e.g., Gln-493 helps with interaction with Glu-35 and Lys-31 on ACE-2). In some embodiments, the spike protein comprises Tyr-505 (e.g., Tyr-505 helps with interaction with Glu-37 and Arg-393 on ACE-2). In some embodiments, the composition comprises a mutation 882-RRAR-885.fwdarw.682-QQAQ-685 in the S1-S2 cleavage site.
[0284] In some embodiments, the composition comprises at least one proline substitution. In some embodiments, the composition comprises at least two proline substitutions. For example, the proline substitution may be at position K988 and V987.
Vaccine Candidates
[0285] As previously discussed, the present invention provides vaccine compositions comprising at least one B cell epitope and at least one CD4+ T cell epitope, at least one B cell epitope and at least one CD8+ T cell epitope, at least one CD4+ T cell epitope and at least one CD8+ T cell epitope, or at least one B cell epitope, at least one CD4+ T cell epitope, and at least one CD8+ T cell epitope.
[0286] In certain embodiments, at least one epitope is derived from a non-spike protein. In certain embodiments, the composition induces immunity to only the epitopes.
[0287] Table 12 and
TABLE-US-00012 TABLE 12 Vaccine SEQ ID Candidate Sequence: NO: 1 CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATT 139 promoter, AGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG 5′UTR and CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA leader CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG sequence, TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA Spike TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG glycoprotein CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC (with 36 TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGC mutations TTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA and 6 TTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCG deletions, CGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG stop CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTC codon, CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC 3′UTR. GCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTC polyA tail CGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTC CCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC GCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTG AGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTG CGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGC CCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCC GCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGC GGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTG GGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCC CTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGG GGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGG TGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGG GGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCT GTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGA GAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCG GCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGC GCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGA CGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTT TTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTT TGGCAAAGAATTGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCA GAGAGAACCCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGA GCAGCCAGTGCGTGAACTTCACCACCAGGACCCAGCTGCCCCCCGCCTA CACCAACAGCTTCACCAGGGGCGTGTACTACCCCGACAAGGTGTTCAGGA GCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCAGCAAC GTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGCACCAAGA GGTTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGC ACCGAGAAGAGCAACATCATCAGGGGCTGGATCTTCGGCACCACCCTGG ACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGT GATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTGT ACTACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCAGGGTGTAC AGCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAGCCCTTCCTGAT GGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGAGGGAGTTCGTG TTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCCATC AACCTGGTGAGGGACCTGCCCCAGGGCTTCAGCGCCCTGGAGCCCCTGG TGGACCTGCCCATCGGCATCAACATCACCAGGTTCCAGACCCTGCTGGCC CTGCACAGGAGCTACCTGACCCCCGGCGACAGCAGCAGCGGCTGGACCG CCGGCGCCGCCGCCTACTACGTGGGCTACCTGCAGCCCAGGACCTTCCT GCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGACTGCGCC CTGGACCCCCTGAGCGAGACCAAGTGCACCCTGAAGAGCTTCACCGTGG AGAAGGGCATCTACCAGACCAGCAACTTCAGGGTGCAGCCCACCGAGAG CATCGTGAGGTTCCCCAACATCACCAACCTGTGCCCCTTCAGCGAGATCT TCAACGCCACCAAGTTCAGCAGCGTGTACGCCTGGGACAGGAGGAAGAT CAACAACTGCGTGGCCGACTACAGCTTCCTGTACAACAGCGCCAGCTTCA GCACCTTCAAGTGCTACGGCGTGAGCCTGAACAAGCTGAACGACCTGTGC TTCACCAACGTGTACGCCGACAGCTTCGTGATCAGGGGCGACCAGGTGAA GCAGATCGCCCCCGGCCAGACCGGCAACATCGCCGACTACAACTACAAG CTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAAGAAGC TGGACAGCAAGGTGGTGGGCAACCACAAGTACAGGTTCAGGTTCTTCAGG AAGAGCAACCTGAAGCCCTTCGAGAGGGACATCAGCACCGAGATCTACCA GGTGGGCAACAAGCCCTGCAAGGGCGCCAAGGGCCTGAACTGCTACCTG CCCCTGAAGAGCTACGGCTTCCAGCCCACCTACGGCGTGGGCTACCAGC CCCACAGGGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCAGCGCCAC CGTGTGCGGCCCCAAGAAGAGCACCAACCTGGTGAAGAACAAGTGCGTG AACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACCGAGAGCA ACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGGGACATCGCCGACACC ACCGACGCCGTGAGGGACCCCCAGACCCTGGAGATCCTGGACATCACCC CCTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCCGGCACCAACACCAG CAACCAGGTGGCCGTGCTGTACCAGGACGTGAACTGCACCGAGGTGCCC GTGGCCATCCACGCCGACCAGCTGACCCCCACCTGGAGGGTGTACAGCA CCGGCAGCAACGTGTTCCAGACCAGGGCCGGCTGCCTGATCGGCGCCGA GCACGTGAACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATC TGCGCCAGCTACCAGACCCAGACCAACAGCCCCAGGAGGGCCAGGAGCG TGGCCAGCCAGAGCATCATCGCCTACACCATGAGCCTGGGCGCCGAGAA CAGCGTGGCCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACCA TCAGCGTGACCACCGAGATCCTGCCCGTGAGCATGACCAAGACCAGCGT GGACTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCAGCAACCTG CTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAACAGGGCCCTGACCG GCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAGGT GAAGCAGATCTACAAGACCCCCCCCATCAAGTACTTCGGCGGCTTCAACT TCAGCCAGATCCTGCCCGACCCCAGCAAGCCCAGCAAGAGGAGCTTCAT CGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTCATC AAGCAGTACGGCGACTGCCTGGGCGACATCGCCGCCAGGGACCTGATCT GCGCCCAGAAGTTCAACGGCCTGACCGTGCTGCCCCCCCTGCTGACCGA CGAGATGATCGCCCAGTACACCAGCGCCCTGCTGGCCGGCACCATCACC AGCGGCTGGACCTTCGGCGCCGGCGCCGCCCTGCAGATCCCCTTCGCCA TGCAGATGGCCTACAGGTTCAACGGCATCGGCGTGACCCAGAACGTGCT GTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCA AGATCCAGGACAGCCTGAGCAGCACCGCCAGCGCCCTGGGCAAGCTGCA GGACGTGGTGAACCAGAACGCCCAGGCCCTGAACACCCTGGTGAAGCAG CTGAGCAGCAACTTCGGCGCCATCAGCAGCGTGCTGAACGACATCCTGA GCAGGCTGGACAAGGTGGAGGCCGAGGTGCAGATCGACAGGCTGATCAC CGGCAGGCTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTGATCAGG GCCGCCGAGATCAGGGCCAGCGCCAACCTGGCCGCCACCAAGATGAGC GAGTGCGTGCTGGGCCAGAGCAAGAGGGTGGACTTCTGCGGCAAGGGCT ACCACCTGATGAGCTTCCCCCAGAGCGCCCCCCACGGCGTGGTGTTCCT GCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACCGCCCCC GCCATCTGCCACGACGGCAAGGCCCACTTCCCCAGGGAGGGCGTGTTCG TGAGCAACGGCACCCACTGGTTCGTGACCCAGAGGAACTTCTACGAGCCC CAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGT GATCGGCATCGTGAACAACACCGTGTACGACCCCCTGCAGCCCGAGCTG GACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCC CGACGTGGACCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAAC ATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAACCTGAACG AGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAA GTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATC GTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCAGCTGCCT GAAGGGCTGCTGCAGCTGCGGCAGCTGCTGCAAGTTCGACGAGGACGAC AGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACC
2 CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATT 140 promoter, ATGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG 5’UTR and CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA leader CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG sequence, TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA Spike TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG glycoprotein CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC (with 36 TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGC mutations TTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA and 6 TTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCG deletions; CGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG 6 CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTC stabilizing CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC mutations), GCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTC stop CGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTC codon, CCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC 3′UTR. GCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTG polyA tail AGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTG CGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGC CCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCC GCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGC GGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTG GGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCC CTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGG GGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGG TGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGG GGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCT GTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGA GAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCG GCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGC GCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGA CGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTT TTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTT TGGCAAAGAATTGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCA GAGAGAACCCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGA GCAGCCAGTGCGTGAACTTCACCACCAGGACCCAGCTGCCCCCCGCCTA CACCAACAGCTTCACCAGGGGCGTGTACTACCCCGACAAGGTGTTCAGGA GCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCAGCAAC GTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGCACCAAGA GGTTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGC ACCGAGAAGAGCAACATCATCAGGGGCTGGATCTTCGGCACCACCCTGG ACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGT GATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTGT ACTACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCAGGGTGTAC AGCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAGCCCTTCCTGAT GGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGAGGGAGTTCGTG TTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCCATC AACCTGGTGAGGGACCTGCCCCAGGGCTTCAGCGCCCTGGAGCCCCTGG TGGACCTGCCCATCGGCATCAACATCACCAGGTTCCAGACCCTGCTGGCC CTGCACAGGAGCTACCTGACCCCCGGCGACAGCAGCAGCGGCTGGACCG CCGGCGCCGCCGCCTACTACGTGGGCTACCTGCAGCCCAGGACCTTCCT GCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGACTGCGCC CTGGACCCCCTGAGCGAGACCAAGTGCACCCTGAAGAGCTTCACCGTGG AGAAGGGCATCTACCAGACCAGCAACTTCAGGGTGCAGCCCACCGAGAG CATCGTGAGGTTCCCCAACATCACCAACCTGTGCCCCTTCAGCGAGATCT TCAACGCCACCAAGTTCAGCAGCGTGTACGCCTGGGACAGGAGGAAGAT CAACAACTGCGTGGCCGACTACAGCTTCCTGTACAACAGCGCCAGCTTCA GCACCTTCAAGTGCTACGGCGTGAGCCTGAACAAGCTGAACGACCTGTGC TTCACCAACGTGTACGCCGACAGCTTCGTGATCAGGGGCGACCAGGTGAA GCAGATCGCCCCCGGCCAGACCGGCAACATCGCCGACTACAACTACAAG CTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAAGAAGC TGGACAGCAAGGTGGTGGGCAACCACAAGTACAGGTTCAGGTTCTTCAGG AAGAGCAACCTGAAGCCCTTCGAGAGGGACATCAGCACCGAGATCTACCA GGTGGGCAACAAGCCCTGCAAGGGCGCCAAGGGCCTGAACTGCTACCTG CCCCTGAAGAGCTACGGCTTCCAGCCCACCTACGGCGTGGGCTACCAGC CCCACAGGGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCAGCGCCAC CGTGTGCGGCCCCAAGAAGAGCACCAACCTGGTGAAGAACAAGTGCGTG AACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACCGAGAGCA ACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGGGACATCGCCGACACC ACCGACGCCGTGAGGGACCCCCAGACCCTGGAGATCCTGGACATCACCC CCTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCCGGCACCAACACCAG CAACCAGGTGGCCGTGCTGTACCAGGACGTGAACTGCACCGAGGTGCCC GTGGCCATCCACGCCGACCAGCTGACCCCCACCTGGAGGGTGTACAGCA CCGGCAGCAACGTGTTCCAGACCAGGGCCGGCTGCCTGATCGGCGCCGA GCACGTGAACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATC TGCGCCAGCTACCAGACCCAGACCAACAGCCCCGGCAGCGCCAGCAGC GTGGCCAGCCAGAGCATCATCGCCTACACCATGAGCCTGGGCGCCGAGA ACAGCGTGGCCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACC ATCAGCGTGACCACCGAGATCCTGCCCGTGAGCATGACCAAGACCAGCG TGGAOTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCAGCAACCT GCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAACAGGGCCCTGACC GGCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAGG TGAAGCAGATCTACAAGACCCCCCCCATCAAGTACTTCGGCGGCTTCAAC TTCAGCCAGATCCTGCCCGACCCCAGCAAGCCCAGCAAGAGGAGCCCCA TCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTCATC AAGCAGTACGGCGACTGCCTGGGCGACATCGCCGCCAGGGACCTGATCT GCGCCCAGAAGTTCAACGGCCTGACCGTGCTGCCCCCCCTGCTGACCGA CGAGATGATCGCCCAGTACACCAGCGCCCTGCTGGCCGGCACCATCACC AGCGGCTGGACCTTCGGCGCCGGCCCCGCCCTGCAGATCCCCTTCCCCA TGCAGATGGCCTACAGGTTCAACGGCATCGGCGTGACCCAGAACGTGCT GTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCA AGATCCAGGACAGCCTGAGCAGCACCCCCAGCGCCCTGGGCAAGCTGCA GGACGTGGTGAACCAGAACGCCCAGGCCCTGAACACCCTGGTGAAGCAG CTGAGCAGCAACTTCGGCGCCATCAGCAGCGTGCTGAACGACATCCTGA GCAGGCTGGACCCCCCCGAGGCCGAGGTGCAGATCGACAGGCTGATCAC CGGCAGGCTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTGATCAGG GCCGCCGAGATCAGGGCCAGCGCCAACCTGGCCGCCACCAAGATGAGC GAGTGCGTGCTGGGCCAGAGCAAGAGGGTGGACTTCTGCGGCAAGGGCT ACCACCTGATGAGCTTCCCCCAGAGCGCCCCCCACGGCGTGGTGTTCCT GCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACCGCCCCC GCCATCTGCCACGACGGCAAGGCCCACTTCCCCAGGGAGGGCGTGTTCG TGAGCAACGGCACCCACTGGTTCGTGACCCAGAGGAACTTCTACGAGCCC CAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGT GATCGGCATCGTGAACAACACCGTGTACGACCCCCTGCAGCCCGAGCTG GACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCC CGACGTGGACCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAAC ATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAACCTGAACG AGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAA GTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATC GTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCAGCTGCCT GAAGGGCTGCTGCAGCTGCGGCAGCTGCTGCAAGTTCGACGAGGACGAC AGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACC
3 CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATT 141 promoter, AGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG 5’UTR and CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA leader CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG sequence, TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA linker, CD TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG 8.sup.+ T cell CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC epitopes, TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGC CD4.sup.+ T TTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA cell TTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCG epitopes, CGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG B Cell CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTc epitopes, CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC stop GCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTC codon, CGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTC 3′UTR, CCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC polyA tail GCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTG AGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTG CGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGC CCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCC GCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGC GGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTG GGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCC CTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGG GGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGG TGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGG GGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCT GTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGA GAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCG GCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGC GCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGA CGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTT TTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTT TGGCAAAGAATTGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCA GAGAGAACCCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGA GCAGCCAGTGCGTGGAGGCCGCCGCCAAGAAGAGCTACGGCTTCCAGCC CACCTACGCCGCCTACGTGGTGGGCAACCACAAGTACAGGTTCGCCGCC TACTACCAGGTGGGCAACAAGCCCTGCAAGGCCGCCTACTGCGTGATCG CCTGGAACAGCAAGAAGGCCGCCTACAAGGGCGCCAAGGGCCTGAACTG CTACGCCGCCTACAGCCAGTGCGTGAACTTCACCACCAGGGCCGCCTAC AACATCGCCGACTACAACTACAAGCTGGCCGCCTACTACCTGCCCCTGAA GAGCTACGGCTTCGCCGCCTACAAGTGCTACGGCGTGAGCCTGAACAAG GCCGCCTACTGCGTGATCGCCTGGAACAGCAAGAAGGCCGCCTACATCTA CAAGACCCCCCCCATCAAGTACGCCGCCTACTGCGTGGCCGACTACAGCT TCCTGTACGCCGCCTACAGCGTGTACGCCTGGGACAGGAGGAAGGCCGC CTACAGGTTCTTCAGGAAGAGCAACCTGAAGGCCGCCTACGACATCAGCA CCGAGATCTACCAGGTGGCCGCCTACTACCAGCCCCACAGGGTGGTGGT GCTGGCCGCCTACGTGGTGGGCAACCACAAGTACAGGTTCGCCGCCTAC TTCGTGATCAGGGGCGACCAGGTGAAGGCCGCCTACAACGCCACCAAGT TCAGCAGCGTGTACGCCGCCTACTACCAGGTGGGCAACAAGCCCTGCAA GGCCGCCTACAACGCCACCAAGTTCAGCAGCGTGTACGCCGCCTACTTC GTGATCAGGGGCGACCAGGTGAAGGCCGCCTACAAGGGCGCCAAGGGC CTGAACTGCTACGCCGCCTACAACCTGTGCCCCTTCAGCGAGATCTTCGC CGCCTACGCCAGCGCCACCGTGGGAAGCGGAGCCACGAACTTCTCTCTG TTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTCAACTGCTACC TGCCCCTGAAGAGCTACGGCTTCCAGCCCACCTACGGCCCCGGCCCCG GCGGCAACCACAAGTACAGGTTCAGGTTCTTCAGGAAGAGCAACCTGG GCCCCGGCCCCGGCCCCTTCGAGAGGGACATCAGCACCGAGATCTACC AGGTGGGCAACGGCCCCGGCCCCGGCAAGAAGCTGGACAGCAAGGTG GTGGGCAACCACAAGTACAGGTTCGGCCCCGGCCCCGGCAAGGGCCTG AACTGCTACCTGCCCCTGAAGAGCTACGGCTTCCAGGGCCCCGGCCCC GGCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAGTGCGTGAACTTCACCG GCCCCGGCCCCGGCAGGGGCGACCAGGTGAAGCAGATCGCCCCCGGC CAGACCGGCAACGGCCCCGGCCCCGGCAGCGCCAGCTTCAGCACCTTC AAGTGCTACGGCGTGAGCCTGAACGGCCCCGGCCCCGGCAAGCTGGAC AGCAAGGTGGTGGGCAACCACAAGTACAGGTTCAGGGGCCCCGGCCCC GGCTTCGCCCAGGTGAAGCAGATCTACAAGACCCCCCCCATCAAGTACG GCCCCGGCCCCGGCGCCGACTACAGCTTCCTGTACAACAGCGCCAGCTT CAGCACCTTCGGCCCCGGCCCCGGCGCCACCAAGTTCAGCAGCGTGTA CGCCTGGGACAGGAGGAAGATCGGCCCCGGCCCCGGCCCCCACAGGG TGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCAGCGGCCCCGGCCCCG GCTTCGAGAGGGACATCAGCACCGAGATCTACCAGGTGGGCAACAAGG GCCCCGGCCCCGGCGCCAAGGGCCTGAACTGCTACCTGCCCCTGAAGA GCTACGGCTTCGGCCCCGGCCCCGGCAGCATCGTGAGGTTCCCCAACAT CACCAACCTGTGCCCCTTCAGCGGCCCCGGCCCCGGCAACAACTGCGT GGCCGACTACAGCTTCCTGTACAACAGCGCCAGCGGCCCCGGCCCCGG CAAGGGCGCCAAGGGCCTGAACTGCTACCTGCCCCTGAAGAGCTACGG CCCCGGCCCCGGCCTGTGCCCCTTCAGCGAGATCTTCAACGCCACCAAG TTCAGCAGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGG AGATGTTGAAGAAAACCCCGGGCCT
AAGAAG
AAGAAG
AAGAAG
AAGAAG
AGAAG
AAGAAG
AAGAAG
AAGAAG
Molecular Adjuvants and T Cell Enhancements
[0288] In certain embodiments, the vaccine composition comprises a molecular adjuvant and/or one or more T Cell enhancement compositions (
[0289] In some embodiments, the vaccine composition comprises a T cell attracting chemokine. The T cell attracting chemokine helps pull the T cells from the circulation to the appropriate tissues, e.g., the lungs, heart, kidney, and brain. Non-limiting examples of T cell attracting chemokines Include CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, or a combination thereof.
[0290] In some embodiments, the vaccine composition comprises a composition that promotes T cell proliferation. Non-limiting examples of compositions that promote T cell proliferation include IL-7, IL-15, IL-2, or a combination thereof.
[0291] In some embodiments, the vaccine composition comprises a composition that promotes T cell homing in the lungs. Non-limiting examples of compositions that promote T cell homing include CCL25, CCL28, CXCL14, CXCL17 or a combination thereof.
[0292] Table 13 shows non-limiting examples of T-cell enhancements that may be used to create a vaccine composition described herein:
TABLE-US-00013 TABLE 13 T-cell SEQ enhancement Sequence ID NO: CXCL11 ATGAACAGGAAGGTGACCGCCATCGCCCTGGCCGCCATCATCTGGGCCA 156 CCGCCGCCCAGGGCTTCCTGATGTTCAAGCAGGGCAGGTGCCTGTGCAT CGGCCCCGGCATGAAGGCCGTGAAGATGGCCGAGATCGAGAAGGCCAG CGTGATCTACCCCAGCAACGGCTGCGACAAGGTGGAGGTGATCGTGACC ATGAAGGCCCACAAGAGGCAGAGGTGCCTGGACCCCAGGAGCAAGCAGG CCAGGCTGATCATGCAGGCCATCGAGAAGAAGAACTTCCTGAGGAGGCA GAACATGTGA CCL5 ATGAAGGTCTCCGCGGCAGCCCTCGCTGTCATCCTCATTGCTACTGCCCT 157 CTGCGCTCCTGCATCTGCCTCCCCATATTCCTCGGACACCACACCCTGCT GCTTTGCCTACATTGCCCGCCCACTGCCCCGTGCCCACATCAAGGAGTAT TTCTACACCAGTGGCAAGTGCTCCAACCCAGCAGTCGTCCACAGGTCAAG GATGCCAAAGAGAGAGGGACAGCAAGTCTGGCAGGATTTCCTGTATGACT CCCGGCTGAACAAGGGCAAGCTTTGTCACCCGAAAGAACCGCCAAGTGT GTGCCAACCCAGAGAAGAAATGGGTTCGGGAGTACATCAACTCTTTGGAG ATGAGCTAGGATGGAGAGTCCTTGAACCTGAACTTACACAAATTTGCCTGT TTCTGCTTGCTCTTGTCCTAGCTTGGGAGGCTTCCCCTCACTATCCTACCC CACCCGCTCCTTGA CXCL9 ATGAAGAAAAGTGGTGTTCTTCCTCTTGGGCATCATCTTGCTGGTTCTG 158 ATTGGAGTGCAAGGAACCCCAGTAGTGAGAAAGGGTCGCTGTTCCTGCAT CAGCACCAACCAAGGGACTATCCACCTACAATCCTTGAAAGACCTTAAACA ATTTGCCCCAAGCCCTTCCTGCGAGAAAATTGAAATCATTGCTACACTGAA GAATGGAGTTCAAACATGTCTAAACCCAGATTCAGCAGATGTGAAGGAACT GATTAAAAAGTGGGAGAAACAGGTCAGCCAAAAGAAAAAGCAAAAGAATG GGAAAAAACATCAAAAAAAGAAAGTTCTGAAAGTTCGAAAATCTCAACGTT CTCGTCAAAAGAAGACTACATAA CXCL10 ATGAATCAAACTGCCATTCTGATTTGCTGCCTTATCTTTCTGACTCTAAGTG 159 GCATTCAAGGAGTACCTCTCTCTAGAACTGTACGCTGTACCTGCATCAGCA TTAGTAATCAACCTGTTAATCCAAGGTCTTTAGAAAAACTTGAAATTATTCC TGCAAGCCAATTTTGTCCACGTGTTGAGATCATTGCTACAATGAAAAAGAA GGGTGAGAAGAGATGTCTGAATCCAGAATCGAAGGCCATCAAGAATTTAC TGAAAGCAGTTAGCAAGGAAAGGTCTAAAAGATCTCCTTAA CXCL14 ATGAGGCTCCTGGCGGCCGCGCTGCTCCTGCTGCTGCTGGCGCTGTACA 160 CCGCGCGTGTGGACGGGTCCAAATGCAAGTGCTCCCGGAAGGGACCCAA GATCCGCTACAGCGACGTGAAGAAGCTGGAAATGAAGCCAAAGTACCCGC ACTGCGAGGAGAAGATGGTTATCATCACCACCAAGAGCGTGTCCAGGTAC CGAGGTCAGGAGCACTGCCTGCACCCCAAGCTGCAGAGCACCAAGCGCT TCATCAAGTGGTACAACGCCTGGAACGAGAAGCGCAGGGTCTACGAAGAA TAG CXCL17 ATGAAAGTTCTAATCTCTTCCCTCCTCCTGTTGCTGCCACTAATGCTGATG 161 TCCATGGTCTCTAGCAGCCTGAATCCAGGGGTCGCCAGAGGCCACAGGG ACCGAGGCCAGGCTTCTAGGAGATGGCTCCAGGAAGGCGGCCAAGAATG TGAGTGCAAAGATTGGTTCCTGAGAGCCCCGAGAAGAAAATTCATGACAG TGTCTGGGCTGCCAAAGAAGCAGTGCCCCTGTGATCATTTCAAGGGCAAT GTGAAGAAAACAAGACACCAAAGGCACCACAGAAAGCCAAACAAGCATTC CAGAGCCTGCCAGCAATTTCTCAAACAATGTCAGCTAAGAAGCTTTGCTCT GCCTTTGTAG CCL25 ATGAACCTGTGGCTCCTGGCCTGCCTGGTGGCCGGCTTCCTGGGAGCCT 162 GGGCCCCCGCTGTCCACACCCAAGGTGTCTTTGAGGACTGCTGCCTGGC CTACCACTACCCCATTGGGTGGGCTGTGCTCCGGCGCGCCTGGACTTAC CGGATCCAGGAGGTGAGCGGGAGCTGCAATCTGCCTGCTGCGATATTCTA CCTCCCCAAGAGACACAGGAAGGTGTGTGGGAACCCCAAAAGCAGGGAG GTGCAGAGAGCCATGAAGCTCCTGGATGCTCGAAATAAGGTTTTTGCAAA GCTCCACCACAACACGCAGACCTTCCAAGCAGGCCCTCATGCTGTAAAGA AGTTGAGTTCTGGAAACTCCAAGTTATCATCGTCCAAGTTTAGCAATCCCA TCAGCAGCAGTAAGAGGAATGTCTCCCTCCTGATATCAGCTAATTCAGGAC TGTGA CCL28 ATGCAGCAGAGAGGACTCGCCATCGTGGCCTTGGCTGTCTGTGCGGCCC 163 TACATGCCTCAGAAGCCATACTTCCCATTGCCTCCAGCTGTTGCACGGAG GTTTCACATCATATTTCCAGAAGGCTCCTGGAAAGAGTGAATATGTGTCGC ATCCAGAGAGCTGATGGGGATTGTGACTTGGCTGCTGTCATCCTTCATGT CAAGCGCAGAAGAATCTGTGTCAGCCCGCACAACCATACTGTTAAGCAGT GGATGAAAGTGCAAGCTGCCAAGAAAAATGGTAAAGGAAATGTTTGCCAC AGGAAGAAACACCATGGCAAGAGGAACAGTAACAGGGCACATCAGGGGA AACACGAAACATACGGCCATAAAACTCCTTATTAG IL-7 ATGTTCCACGTGAGCTTCAGGTACATCTTCGGCATCCCCCCCCTGATCCT 164 GGTGCTGCTGCCCGTGACCAGCAGCGAGTGCCACATCAAGGACAAGGAG GGCAAGGCCTACGAGAGCGTGCTGATGATCAGCATCGACGAGCTGGACA AGATGACCGGCACCGACAGCAACTGCCCCAACAACGAGCCCAACTTCTTC AGGAAGCACGTGTGCGACGACACCAAGGAGGCCGCCTTCCTGAACAGGG CCGCCAGGAAGCTGAAGCAGTTCCTGAAGATGAACATCAGCGAGGAGTTC AACGTGCACCTGCTGACCGTGAGCCAGGGCACCCAGACCCTGGTGAACT GCACCAGCAAGGAGGAGAAGAACGTGAAGGAGCAGAAGAAGAACGACGC CTGCTTCCTGAAGAGGCTGCTGAGGGAGATCAAGACCTGCTGGAACAAGA TCCTGAAGGGCAGCATCTGA IL-15 ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGT 165 GTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCAT TTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGA ATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATT GATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACA GCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGA GATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAAC AGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAG GAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTG TCCAAATGTTCATCAACACTTCTTGA IL-2 ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTC 166 ACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTG GAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACA AGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGA AGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTC TGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCA GGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTG AAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAAT TTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTG A
[0293] In some embodiments, the T-cell enhancement compositions described herein (e.g. CXCL9, CXCL10, IL-7, IL-2) may be integrated Into a separate delivery system from the vaccine compositions. In other embodiments, the T-cell enhancement compositions described herein (e.g. CXCL9, CXCL10, IL-7, IL-2) may be integrated into the same delivery system as the vaccine compositions.
[0294] In certain embodiments, the composition comprises a tag. For example, in some embodiments, the composition comprises a His tag. The present invention is not limited to a His tag and Includes other tags such as those known to one of ordinary skill in the art, such as a fluorescent tag (e.g., GFP, YFP, etc.), etc.
Antigen Delivery System
[0295] The present invention also features vaccine compositions in the form of an antigen delivery system. Any appropriate antigen delivery system may be considered for delivery of the antigens described herein. The present invention is not limited to the antigen delivery systems described herein.
[0296] In certain embodiments, the antigen delivery system is for targeted delivery of the vaccine composition, e.g., for targeting to the tissues of the body where the virus replicates.
[0297] In certain embodiments, the antigen delivery system comprises an adeno-associated virus vector-based antigen delivery system, such as but not limited to the adeno-associated virus vector type 9 (AAV9 serotype), AAV type 8 (AAV8 serotype), etc. (see, for example,
[0298] The present invention is not limited to adeno-associated virus vector-based antigen delivery systems. Examples of other antigen delivery systems include: adenoviruses such as but not limited to Ad5, Ad26, Ad35, etc., as well as carriers such as lipid nanoparticles, polymers, peptides, etc. In other embodiments, the antigen delivery system comprises a vesicular stomatitis virus (VSV) vector.
[0299] In the antigen delivery system, the antigen or antigens (e.g., epitopes) are operatively linked to a promoter. In certain embodiments, the antigen or antigens (e.g., epitopes) are operatively linked to a generic promoter. For example, in certain embodiments, the antigen or antigens (e.g., epitopes) are operatively linked to a CMV promoter. In certain embodiments, the antigen or antigens (e.g., epitopes) are operatively linked to a CAG, EFIA, EFS, CBh, SFFV, MSCV, mPGK, hPGK, SV40, UBC, or other appropriate promoter.
[0300] In some embodiments, the antigen or antigens (e.g., epitopes) are operatively linked to a tissue-specific promoter (e.g., a lung-specific promoter). For example, the antigen or antigens (e.g., epitopes) are may be operatively linked to a SpB promoter or a CD144 promoter.
[0301] As discussed, in certain embodiments, the vaccine composition comprises a molecular adjuvant. In certain embodiments, the molecular adjuvant is operatively linked to a generic promoter, e.g., as described above. In certain embodiments, the molecular adjuvant is operatively linked to a tissue-specific promoter, e.g., a lung-specific promoter, e.g., SpB or CD144 (see
[0302] As discussed, in certain embodiments, the vaccine composition comprises a T cell attracting chemokine. In certain embodiments, the T cell attracting chemokine is operatively linked to a generic promoter, e.g., as described above. In certain embodiments, the T cell attracting chemokine is operatively linked to a tissue-specific promoter, e.g., a lung-specific promoter, e.g., SpB or CD144 (e.g., see
[0303] As discussed, in certain embodiments, the vaccine composition comprises a composition for promoting T cell proliferation. In certain embodiments, the composition for promoting T cell proliferation is operatively linked to a generic promoter, e.g., as described above. In certain embodiments, the composition for promoting T cell proliferation is operatively linked to a tissue-specific promoter, e.g., a lung-specific promoter, e.g., SpB or CD144 (e.g., see
[0304] Table 14 shows non-limiting examples of promoters that may be used to create a vaccine composition described herein.
TABLE-US-00014 TABLE 14 SEQ Promoter Sequence ID NO: CAG CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT 167 CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTA AACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGG GGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGG GCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCC GAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGA AGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCT CCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCA CAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGT TTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCG GGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTG TGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCT GCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCG CGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAG GCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGT CGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGC CCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGC CGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCT CGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGG CGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCG AGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGG AGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCC GGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTC CCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGG GGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTA GAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAA CGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTG CMV TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGT 168 TCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGA CTTTOCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAA TGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACC CCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAA ATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTG GGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC SP-B GTATAGGGCTGTCTGGGAGCCACTCCAGGGCCACAGAAATCTTGTCTCTGACTC 169 AGGGTATTTTGTTTTCTGTTTTGTGTAAATGCTCTTCTGACTAATGCAAACCATGT GTCCATAGAACCAGAAGATTTTTCCAGGGGAAAAGGTAAGGAGGTGGTGAGAGT GTCCTGGGTCTGCCCTTCCAGGGCTTGCCCTGGGTTAAGAGCCAGGCAGGAAG CTCTCAAGAGCATTGCTCAAGAGTAGAGGGGGCCTGGGAGGCCCAGGGAGGG GATGGGAGGGGAACACCCAGGCTGCCCCCAACCAGATGCCCTCCACCCTCCTC AACCTCCCTCCCACGGCCTGGAGAGGTGGGACCAGGTATGGAGGCTTGAGAGC CCCTGGTTGGAGGAAGCCACAAGTCCAGGAACATGGGAGTCTGGGCAGGGGGC AAAGGAGGCAGGAACAGGCCATCAGCCAGGACAGGTGGTAAGGCAGGCAGGA GTGTTCCTGCTGGGAAAAGGTGGGATCAAGCACCTGGAGGGCTCTTCAGAGCA AAGACAAACACTGAGGTCGCTGCCACTCCTACAGAGCCCCCACGCCCCGCCCA GCTATAAGGGGCCATGCACCAAGCAGGGTACCCAGGCTGCAGAGGTGCC CD144 CATCCATGCCCATGGCCTCAGATGCCAGCCATAAGCTGTTGGGTTCCAAACCTC 170 GACTCCAGGCTGGACTCACCCCTGTCTCCCCCACCAGCCTGACACCTCCACCTG GGTATCTAACGAGCATCTCAAACTCAACCTGCCTGAGACAGAGGAATCACTATCC CCTCCTCCTCCAAAAATATCCTTCCATCACACTCCCCATCTTGTGCTCTGATTTAC TAAACGGCCCTGGGCCCTCTCTTTCTCAGGGTCTCTGCTTGCCCAGCTATATAAT AAAACAAGTTTGGGACTTCCCAACCATTCACCCATGGAAAAACAGAAGCAACTCT TCAAAGGACAGATTCCCAGGATCTGCCCTGGGAGATTCCAAATCAGTTGATCTG GGGTGAGCCCAGTCCTCTGTAGTTTTTAGAAGCTCCTCCTATGTCTCTCCTGGTC AGCAGAATCTTGGCCCCTCCCTTCCCCCCAGCCTCTTGGTTCTTCTGGGCTCTG ATCCAGCCTCAGCGTCACTGTCTTCCACGCCCCTCTTTGATTCTCGTTTATGTCA AAAGCCTTGTGAGGATGAGGCTGTGATTATCCCCATTTTACAGATGAGGAAACTG TGGCTCCAGGATGACACAACTGGCCAGAGGTCACATCAGAAGCAGAGCTGGGT CACTTGACTCCACCCAATATCCCTAAATGCAAACATCCCCTACAGACCGAGGCTG GCACCTTAGAGCTGGAGTCCATGCCCGCTCTGACCAGGAGAAGCCAACCTGGT CCTCCAGAGCCAAGAGCTTCTGTCCCTTTCCCATCTCCTGAAGCCTCCCTGTCA CCTTTAAAGTCCATTCCCACAAAGACATCATGGGATCACCACAGAAAATCAAGCT CTGGGGCTAGGCTGACCCCAGCTAGATTTTTGGCTCTTTTATACCCCAGCTGGG TGGACAAGCACCTTAAACCCGCTGAGCCTCAGCTTCCCGGGCTATAAAATGGGG GTGATGACACCTGCCTGTAGCATTCCAAGGAGGGTTAAATGTGATGCTGCAGCC AAGGGTCCCCACAGCCAGGCTCTTTGCAGGTGCTGGGTTCAGAGTCCCAGAGC TGAGGCCGGGAGTAGGGGTTCAAGTGGGGTGCCCCAGGCAGGGTCCAGTGCC AGCCCTCTGTGGAGACAGCCATCCGGGGCCGAGGCAGCCGCCCACCGCAGGG CCTGCCTATCTGCAGCCAGCCCAGCCCTCACAAAGGAACAATAACAGGAAACCA TCCCAGGGGGAAGTGGGCCAGGGCCAGCTGGAAAACCTGAAGGGGAGGCAGC CAGGCCTCCCTCGCCAGCGGGGTGTGGCTCCCCTCCAAAGACGGTCGGCTGAC AGGCTCCACAGAGCTCCACTCACGCTCAGCCCTGGACGGACAGGCAGTCCAAC GGAACAGAAACATCCCTCAGCCCACAGGCACGGTGAGTGGGGGCTCCCACACT CCCCTCCACCCCAAACCCGCCACCCTGCGCCCAAGATGGGAGGGTCCTCAGCT TCCCCATCTGTAGAATGGGCATCGTCCCACTCCCATGACAGAGAGGCTCC wild type ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGC 171 native leader sequence
[0305] In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter (e.g., the T cell attracting chemokine and the composition that promotes T cell proliferation are synthesized as a peptide). In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by different promoters. In certain embodiments, the antigen, the T cell attracting chemokine, and the composition that promotes T cell proliferation are driven by the same promoter. In certain embodiments, the antigen or antigens, the T cell attracting chemokine, and the composition that promotes T cell proliferation are driven by the different promoters. In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter, and the antigen or antigens are driven by a different promoter.
[0306] In some embodiments, the antigen delivery system comprises one or more linkers between the T cell attracting chemokine and the composition that promotes T cell proliferation. In certain embodiments, linkers are used between one or more of the epitopes. The linkers may allow for cleavage of the separate molecules (e.g., chemokine). For example, in some embodiments, a linker is positioned between IL-7 (or IL-2) and CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, etc. In some embodiments, a linker is positioned between IL-15 and CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, etc. In some embodiments, a linker is positioned between the antigen and another composition, e.g., IL-15, IL-7, IL-2, CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, etc. A non-limiting example of a linker is T2A, E2A, P2A (see Table 15), or the like (e.g., see
TABLE-US-00015 TABLE 15 SEQ SEQUENCE ID NO: T2A Linker GGAAGCGGAGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGG 142 AGGAAAATCCCGGCCCC E2A Linker GGAAGCGGACAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGAT 152 GTTGAGAGCAACCCAGGTCCC P2A Linker GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGT 180 TGAAGAAAACCCCGGGCCT 6-His Tag CATCACCATCACCATCAC 181
[0307] The present invention includes mRNA sequences encoding any of the vaccine compositions or portions thereof herein. The present invention also includes modified mRNA sequences encoding any of the vaccine compositions or portions thereof herein. The present invention also includes DNA sequence encoding any of the vaccine compositions or portions thereof herein.
[0308] In certain embodiments, nucleic acids of a vaccine composition herein are chemically modified. In some embodiments, the nucleic acids of a vaccine composition therein are unmodified. In some embodiments, all or a portion of the uracil in the open reading frame has a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1-methyl pseudouridine. In some embodiments, all or a portion of the uracil in the open reading frame has a N1-methyl pseudouridine in the 5-position of the uracil.
[0309] In certain embodiments, an open reading frame of a vaccine composition herein encodes one antigen or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes two or more antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes five or more antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes ten or more antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes 50 or more antigens or epitopes.
Epitope Arrangements
[0310] The target epitopes of the compositions described may be arranged in various configurations (see, for example,
[0311] In some embodiments, the target epitopes may be arranged such that one or more pairs of CD4+-CD8+ T cell epitopes are followed by one or more pairs of CD4+ T cell-B cell epitopes. In other embodiments, the target epitopes may be arranged such that CD8+ T cell, CD4+ T cell, and B cell epitopes are repeated one or more times.
[0312] In other embodiments, the target epitopes may be arranged such that one or more CD4+ T cell epitopes are followed by one or more CD8+ T cell epitopes. In embodiments, the target epitopes may be arranged such that one or more CD8+ T cell epitopes are followed by one or more CD4+ T cell epitopes. In some embodiments, the target epitopes may be arranged such that one or more CD4+ T cell epitopes are followed by one or more B cell target epitopes. In some embodiments, the target epitopes may be arranged such that one or more CD8+ T cell epitopes are followed by one or more B cell target epitopes. In other embodiments, the target epitopes may be arranged such that one or more B cell epitopes are followed by one or more CD4+ T cell target epitopes. In some embodiments, the target epitopes may be arranged such that one or more B cell epitopes are followed by one or more CD8+ T cell target epitopes.
[0313] Likewise, the other components of the vaccine composition may be arranged in various configurations. For example, in certain embodiments, the T cell attracting chemokine is followed by the composition for promoting T cell proliferation. In certain embodiments, the composition for promoting T cell proliferation is followed by the T cell attracting chemokine.
Methods
[0314] The present invention also features methods for designing and/or producing a pan-coronavirus composition. Briefly, the method may comprise determining target epitopes, selecting desired target epitopes (e.g., two or more, etc.), and synthesizing an antigen comprising the selected target epitopes. The method may comprise determining target epitopes, selecting desired target epitopes, and synthesizing a nucleotide composition (e.g., DNA, modified DNA, mRNA, modified mRNA, antigen delivery system, etc.) encoding the antigen comprising the selected target epitopes. In some embodiments, the method further comprises creating a vaccine composition comprising the antigen, nucleotide compositions, and/or antigen delivery system and a pharmaceutical carrier.
[0315] The methods herein may also include the steps of designing the antigen delivery system. For example, the methods may comprise inserting molecular adjuvants, chemokines, linkers, tags, etc. into the antigen delivery system. In some embodiments, one or more components is inserted into a different antigen delivery system from the antigen or antigens (e.g., the epitopes). For example, the present invention provides embodiments wherein the antigen or antigens (e.g., the epitopes) are within a first antigen delivery system and one or more additional components (e.g., chemokine, etc.) are within a second delivery system. In some embodiments, the antigen or antigens (e.g., the epitopes) and one or more additional components are within a first delivery system, and one or more additional components are within a second delivery system. In some embodiments, the antigen or antigens (e.g., the epitopes) and one or more additional components are within a first delivery system, and the antigen or antigens (e.g., the epitopes) and one or more additional components are within a second delivery system.
[0316] In some embodiments, the method comprises determining target epitopes from at least two of the following 1. coronavirus B-cell epitopes, 2. coronavirus CD4+ T cell epitopes, and/or 3. coronavirus CD8+ T cell epitopes. In some embodiments, each of the target epitopes are mutated epitopes, e.g., as described herein. For example, the target epitopes may be mutated among two or a combination of at least one SARS-CoV-2 human strains in current circulation, at least one coronavirus that has caused a previous human outbreak, at least one coronavirus isolated from bats, at least one coronavirus Isolated from pangolin, at least one coronavirus isolated from civet cats, at least one coronavirus strain isolated from mink, and at least one coronavirus strain isolated from camels or any other animal that is receptive to coronavirus. In some embodiments, the composition comprises at least two of the following: one or more coronavirus B-cell target epitopes, one or more coronavirus CD4.sup.+ T cell target epitopes, and/or one or more coronavirus CD8.sup.+ T cell target epitopes.
[0317] In certain embodiments, the method comprises selecting at least one epitope from at least two of: one or more mutated coronavirus B-cell epitopes; one or more mutated coronavirus CD4+ T cell epitopes; and one or more mutated coronavirus CD8+ T cell epitopes: and synthesizing an antigen comprising the selected epitopes. In certain embodiments, the method comprises selecting at least one epitope from at least two of: one or more mutated coronavirus B-cell epitopes; one or more mutated coronavirus CD4+ T cell epitopes; and one or more mutated coronavirus CD8+ T cell epitopes; and synthesizing an antigen delivery system that encodes an antigen comprising the selected epitopes.
[0318] In some embodiments, the method comprises determining one or more mutated large sequences that are derived from coronavirus sequences (e.g., SARS-CoV-2, variants, common cold coronaviruses, previously known coronavirus strains, animal coronaviruses, etc.). The method may comprise selecting at least one large mutated sequence and synthesizing an antigen comprising the selected large mutated sequence(s). The method may comprise synthesizing a nucleotide composition (e.g., DNA, modified DNA. mRNA, modified mRNA, antigen delivery system, etc.) encoding the antigen comprising the selected large mutated sequence(s). In some embodiments, the method further comprises creating a vaccine composition comprising the antigen, nucleotide compositions, and/or antigen delivery system and a pharmaceutical carrier. In some embodiments, the large sequences comprise one or more mutated epitopes described herein, e.g., one or more mutated B-cell target epitopes and/or one or more mutatedCD4+ T cell target epitopes and/or one or more mutatedCD8+ T cell target epitopes.
[0319] In some embodiments, each of the large sequences are mutated among two or a combination of: at least two SARS-CoV-2 human strains in current circulation, at least one coronavirus that has caused a previous human outbreak, at least one coronavirus isolated from bats, at least one coronavirus isolated from pangolin, at least one coronavirus Isolated from civet cats, at least one coronavirus strain isolated from mink, and at least one coronavirus strain isolated from camels or any other animal that is receptive to coronavirus.
[0320] As previously discussed, the compositions described herein, e.g., the epitopes, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc. may be used to prevent a coronavirus disease in a subject. In some embodiments, the compositions described herein, e.g., the antigen or antigens (e.g., epitopes), the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc. may be used to prevent a coronavirus infection prophylactically in a subject. In some embodiments, the compositions described herein, e.g., the epitopes, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc. may elicit an immune response in a subject. In some embodiments, the compositions described herein, e.g., the epitopes, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc. may prolong an immune response induced by the multi-epitope pan-coronavirus vaccine composition and increases T-cell migration to the lungs.
[0321] Methods for preventing a coronavirus disease in a subject may comprise administering to the subject a therapeutically effective amount of a pan-coronavirus vaccine composition according to the present invention. In some embodiments, the composition elicits an immune response in the subject. In some embodiments, the composition induces memory B and T cells. In some embodiments, the composition induces resident memory T cells (T.sub.rm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition prevents inflammation or an inflammatory response. e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition improves homing and retention of T cells, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney.
[0322] Methods for preventing a coronavirus infection prophylactically in a subject may comprise administering to the subject a prophylactically effective amount of a pan-coronavirus vaccine composition according to the present invention. In some embodiments, the composition elicits an immune response in the subject. In some embodiments, the composition induces memory B and T cells. In some embodiments, the composition induces resident memory T cells (Trm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition prevents inflammation or an inflammatory response, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition Improves homing and retention of T cells, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney.
[0323] Methods for eliciting an immune response in a subject may comprise administering to the subject a vaccine composition according to the present invention, wherein the composition elicits an immune response in the subject. In some embodiments, the composition induces memory B and T cells. In some embodiments, the composition induces resident memory T cells (Trm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition prevents Inflammation or an inflammatory response, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney. In some embodiments, the composition improves homing and retention of T cells, e.g., in the areas where the virus normally replicates such as lungs, brain, heart, and kidney.
[0324] Methods for prolonging an immune response induced by a vaccine composition of the present invention and increasing T cell migration to particular tissues (e.g., lung, brain, heart, kidney, etc.) may comprise co-expressing a T-cell attracting chemokine, a composition that promotes T cell proliferation, and a vaccine composition (e.g., antigen) according to the present invention.
[0325] Methods for prolonging the retention of memory T-cell into the lungs induced by a vaccine composition of the present invention and increasing virus-specific tissue resident memory T-cells (TRM cells) may comprise co-expressing a T-cell attracting chemokine, a composition that promotes T cell proliferation, and a vaccine composition (e.g., antigen) according to the present invention.
[0326] The vaccine composition may be administered through standard means, e.g., through an intravenous route (i.v.), an Intranasal route (i.n.), or a sublingual route (s.l.) route.
[0327] In certain embodiments, the method comprises administering to the subject a second (e.g., booster) dose. The second dose may comprise the same vaccine composition or a different vaccine composition. Additional doses of one or more vaccine compositions may be administered.
Sequential Vaccine Delivery Methodology
[0328] In some embodiments, the present invention features a method of delivering the vaccine to induce heterologous immunity in a subject (e.g., prime/boost, see
[0329] In some embodiments, the first delivery system or the second delivery system comprises an mRNA, a modified mRNA or a peptide vector. In other embodiments, the peptide vector comprises adenovirus or an adeno-associated virus vector.
[0330] In some embodiments, the present invention features a method of delivering the vaccine to induce heterologous immunity in a subject (e.g., prime/pull, see
[0331] The present invention also features a novel “prime, pull, and boost” strategy. In other embodiments, the present invention features a method to increase the size and maintenance of lung-resident B-cells, CD4+ T cells and CD8+ T cells to protect against SARS-CoV-2 (
[0332] The present invention further features a novel “prime, pull, and keep” strategy (
[0333] In some embodiments, the mucosal chemokines may comprise CCL25, CCL28, CXCL14, CXCL17, or a combination thereof. In some embodiments, the T-cell attracting chemokines may comprise CCL5, CXCL9, CXCL10, CXCL11, or a combination thereof. In some embodiments, the cytokines may comprise IL-15, IL-7, IL-2, or a combination thereof.
[0334] In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 60%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 70%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 80%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 90%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 95%.
[0335] Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11):1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas: Efficacy=(ARU−ARV)/ARUx100; and Efficacy=(1−RR)×100.
[0336] Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11):1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness=(1−OR)×100.
[0337] In some embodiments, the vaccine immunizes the subject against a coronavirus for up to 1 year. In some embodiments, the vaccine immunizes the subject against a coronavirus for up to 2 years. In some embodiments, the vaccine immunizes the subject against a coronavirus for more than 1 year, more than 2 years, more than 3 years, more than 4 years, or for 5-10 years.
[0338] In some embodiments, the subject is a young adult between the ages of about 20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45 or 50 years old).
[0339] In some embodiments, the subject is an elderly subject about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85 or 90 years old).
[0340] In some embodiments, the subject is about 5 years old or younger. For example, the subject may be between the ages of about 1 year and about 5 years (e.g., about 1, 2, 3, 5 or 5 years), or between the ages of about 6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months). In some embodiments, the subject is about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month). In some embodiments, the subject is about 6 months or younger.
[0341] In some embodiments, the subject was born full term (e.g., about 37-42 weeks). In some embodiments, the subject was born prematurely, for example, at about 36 weeks of gestation or earlier (e.g., about 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 or 25 weeks). For example, the subject may have been born at about 32 weeks of gestation or earlier. In some embodiments, the subject was born prematurely between about 32 weeks and about 36 weeks of gestation. In such subjects, a vaccine may be administered later in life, for example, at the age of about 6 months to about 5 years, or older.
[0342] In some embodiments, the subject is pregnant (e.g., in the first, second or third trimester) when administered a vaccine.
[0343] In some embodiments, the subject has a chronic pulmonary disease (e.g., chronic obstructive pulmonary disease (COPD) or asthma) or is at risk thereof. Two forms of COPD include chronic bronchitis, which involves a long-term cough with mucus, and emphysema, which involves damage to the lungs over time. Thus, a subject administered a vaccine may have chronic bronchitis or emphysema.
[0344] In some embodiments, the subject has been exposed to a coronavirus. In some embodiments, the subject is infected with a coronavirus. In some embodiments, the subject is at risk of infection by a coronavirus.
[0345] In some embodiments, the subject is immunocompromised (has an impaired immune system, e.g., has an immune disorder or autoimmune disorder).
Pharmaceutical Carriers
[0346] In certain embodiments, the vaccine composition further comprises a pharmaceutical carrier. Pharmaceutical carriers are well known to one of ordinary skill in the art. For example, in certain embodiments, the pharmaceutical carrier is selected from the group consisting of water, an alcohol, a natural or hardened oil, a natural or hardened wax, a calcium carbonate, a sodium carbonate, a calcium phosphate, kaolin, talc, lactose and combinations thereof. In some embodiments, the pharmaceutical carrier may comprise a lipid nanoparticle, an adenovirus vector, or an adeno-associated virus vector. In some embodiments, the vaccine composition is constructed using an adeno-associated virus vectors-based antigen delivery system.
[0347] Also provided herein is vaccine of any one of the foregoing paragraphs, formulated in a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoy)oxy)heptadecanedioate (L319).
[0348] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.